WO2010022145A2 - Alternatively-spliced isoform of mu-opioid receptor gene with cell excitatory function - Google Patents

Alternatively-spliced isoform of mu-opioid receptor gene with cell excitatory function Download PDF

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WO2010022145A2
WO2010022145A2 PCT/US2009/054300 US2009054300W WO2010022145A2 WO 2010022145 A2 WO2010022145 A2 WO 2010022145A2 US 2009054300 W US2009054300 W US 2009054300W WO 2010022145 A2 WO2010022145 A2 WO 2010022145A2
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polypeptide
seq
nucleic acid
opioid receptor
sequence
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WO2010022145A3 (en
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Luda Diatchenko
William Maixner
Pavel Gris
Josee Gauthier
Andrea Nackley
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The University Of North Carolina At Chapel Hill
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9486—Analgesics, e.g. opiates, aspirine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways

Definitions

  • TECHNICAL FIELD The presently disclosed subject matter describes a class of splice variant forms of the ⁇ -opioid receptor having cell excitatory characteristics. Also described are methods of screening drug candidates to find compounds that provide analgesia free of hyperalgesia-like effects.
  • the mu( ⁇ )-type opioid receptor (O PRM 1) is a member of the G-protein- coupled receptor (GPCR) family. It has an extracellular N-terminus and intracellular C-terminus, with seven membrane-spanning domains that comprise the binding pocket for exogenous drugs. Upon activation, these seven transmembrane (7TM) domain GPCRs initiate molecular changes resulting in inhibition of nerve, immune, and glial cells that play a role in the onset and maintenance of pain.
  • GPCR G-protein- coupled receptor
  • OPRM1 induces analgesia via pertussis toxin (PTX)-sensitive inhibitory G protein (G ⁇ j /0 ), which inhibits cAMP formation and Ca 2+ conductance and activates K + conductance, leading to hyper-polarization of cells thereby, exerting an inhibitory effect.
  • PTX pertussis toxin
  • G ⁇ j /0 pertussis toxin-sensitive inhibitory G protein
  • OPRM1 induces analgesia via pertussis toxin (PTX)-sensitive inhibitory G protein (G ⁇ j /0 ), which inhibits cAMP formation and Ca 2+ conductance and activates K + conductance, leading to hyper-polarization of cells thereby, exerting an inhibitory effect.
  • PTX pertussis toxin
  • G ⁇ j /0 pertussis toxin
  • opioids Prolonged use of opioids leads to a number of adverse side-effects including constipation, CNS depression, dependence and potentially the exacerbation of pain via a phenomenon commonly referred to as "opioid-induced hyperalgesia" (OIH).
  • OIH opioid-induced hyperalgesia
  • MOR agonists MOR agonists
  • Both extremely low and extremely high doses of morphine, as well as chronic administration of opioids can elicit a hyperalgesia in animal models of pain. See Crain and Shen, Brain Res., 888, 75-82 (2001); and Li et al.. Brain Res. MoI. Brain Res., 86, 56-62 (2001).
  • OPRM1 also called MOR-1 binds endogenous and exogenous opioids to produce analgesia, and to mediate basal nociception as well as agonist responses
  • MOR-1 binds endogenous and exogenous opioids to produce analgesia, and to mediate basal nociception as well as agonist responses
  • MOR-1 is coded by exons 1 , 2, 3 and 4, whereas exon 1 codes for first transmembrane domain and exon 2 and 3 code for the second through the seventh transmembrane domain. See Pasternak, Neuropharmacology, 47 Suppl 1, 312-323 (2004). There is growing evidence from rodent studies that could suggest a role of alternatively-spliced forms of OPRM1 in mediating opiate analgesia.
  • mouse OPRM1 gene includes 20 exons and codes for 41 alternative-spliced forms, while the human OPRM 1 gene includes only 9 exons and codes for only 19 alternative-spliced forms.
  • C-terminus variants contain exons 1 , 2 and 3 and code for all seven transmembrane domains, but differ structurally and functionally at the intracellular domain, a region important in transduction of the signal following receptor activation.
  • the mouse also has a number of variants that differ in their N-terminus, some of which encode for truncated receptors.
  • Reported mouse N-terminus variants are initiated from exon 11. Exon 11 is located approximately 10 kb upstream of exon 1 and is under the control of a different upstream promoter, suggesting alternative regulation of transcription.
  • Three of these variants are predicted to code for truncated receptors with only six transmembrane domains (6TM), lacking TM1.
  • This isoform lacks an amino acid sequence of approximately 90 amino acids that constitutes the extracellular N-terminal and first transmembrane TMH 1 domains and part of the first intracellular loop, but retains the ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains. Additionally, the receptor proteins are terminated by intracellular C-terminal amino acid sequences that have been hypothesized to serve as coupling or docking domains required for constitutive NO synthase (NOS) activation, which is unique among OPRM 1 isoforms. See Kream, et al., Med. Sci. Monit, 13, SC5-SC6 (2007).
  • NOS NO synthase
  • the presently disclosed subject matter provides a class of splice variant forms of human OPRM1 and polynucleotide sequences encoding the OPRM1 splice variants. More particularly, the presently disclosed subject matter relates to exon-13 containing 6TM MOR-1 K isoforms of human OPRM1 , including alternative splice variants MOR-1 K1 and MOR-1 K2.
  • an isolated nucleic acid sequence consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 1 , 2 or a nucleic acid sequence having at least a 98% sequence homology to SEQ ID NO: 1 or 2 over its entire length is provided.
  • an isolated nucleic acid sequence which codes for a protein consisting essentially of an amino acid sequence of
  • SEQ ID NO: 3 or for a protein comprising amino acids 1-300 of SEQ ID NO: 3.
  • inventions of the presently disclosed subject matter include a recombinant vector or a recombinant host cell comprising any of the nucleic acid sequences described above.
  • the presently disclosed subject matter provides a nucleic acid sequence that is at least 97% identical to a contiguous 902 nucleotide sequence selected from nucleotides 1309-2211 of SEQ ID NO: 1 or nucleotides 1290-2192 of SEQ ID NO: 2.
  • Another embodiment provides a substantially pure polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 3 or a sequence having a 97% or greater sequence homology to SEQ ID NO: 3 over its entire length; or of amino acids 1-300 of SEQ ID NO: 3 or a sequence having at least a 97% sequence homology to the sequence comprising amino acids 1-300 of SEQ ID NO: 3 over its entire length.
  • the presently disclosed subject matter includes a recombinant cell expressing any of the polypeptides described above.
  • the presently disclosed subject matter further relates to methods of screening compounds for their ability to regulate the activity of MOR-1 splice variants.
  • a method of screening candidate substances for an ability to modulate activity of a six-transmembrane ⁇ -opioid receptor isoform comprises providing a test sample comprising any or the polypeptides described above, administering a test molecule to the test sample, and determining the effect of the test molecule on the activity of the polypeptide.
  • the test molecule is selected from the group consisting of a polypeptide, a nucleic acid oligonucleotide, an exogenous vector coding for a nucleic acid oligonucleotide or polypeptide, a carbohydrate, a lipid, an amino acid, a fatty acid, a steroid, and a low molecular weight organic molecule.
  • determining the effect of the test molecule on the activity of the polypeptide comprises measuring a first activity level of the polypeptide prior to administering the test molecule to the test sample, measuring a second activity level of the polypeptide after administering the test molecule to the test sample, and comparing the first and second activity levels.
  • the above method further comprises determining the effect of the test molecule on the activity of a polypeptide comprising the sequence of SEQ ID NO: 4 or amino acids 67-330 of SEQ ID NO: 4 and comparing said effect with the effect of the test molecule on the activity of any of the polypeptides of the embodiments described above.
  • the test sample is a cell.
  • the presently disclosed subject matter includes methods wherein the polypeptide is provided to the cell from an exogenous source or wherein the cell expresses the polypeptide.
  • the cell is a recombinant cell.
  • determining the effect of the test molecule comprises measuring an amount or a change in the amount of one or more of the group consisting of cAMP, calcium, nitric oxide or coupling to G ⁇ s protein in the test sample.
  • the test sample is a non-human animal.
  • the presently disclosed subject matter includes methods where determining the effect of the test compound is estimated by assessment of pain-related behavior in the animal. Also included are methods wherein the animal is a genetically modified animal.
  • the genetically modified animal may possess modulated six transmembrane ⁇ -opioid receptor isoform activity, modulated seven transmembrane ⁇ -opioid receptor isoform activity, or a combination thereof.
  • the transgenic animal overexpresses any of the polypeptide embodiments described above.
  • the genetically modified animal may be a knock-out animal that under expresses a seven transmembrane ⁇ -opioid receptor isoform.
  • the method includes cases where the under-expressed 7TM isoform is a polypeptide comprising the sequence of SEQ ID NO: 4 or amino acids 67-330 of SEQ ID NO: 4.
  • a transgenic non-human animal wherein the transgenic non-human animal overexpresses a six-transmembrane ⁇ -opioid receptor isoform comprising a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology to amino acids 1-300 of SEQ ID NO: 3 over the entire length of the amino acid sequence.
  • a method for identifying an antagonist or an agonist specific for a six transmembrane ⁇ -opioid receptor isoform wherein the isoform comprises a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology thereto over the entire length of the amino acid, the method comprising contacting a cell with a test molecule and a ⁇ - opioid receptor agonist or antagonist, wherein said cell comprises a nucleic acid molecule consisting essentially of one of SEQ ID NOs: 1 or 2 or a sequence having at least a 98% homology thereto, and wherein said cell expresses said polypeptide; and determining whether or not said test molecule reduces or prevents a non-analgesic or hyperanalgesic ⁇ -opioid receptor- mediated response induced by said ⁇ -opioid receptor agonist or antagonist.
  • a method of producing an antibody immunoreactive with a six transmembrane ⁇ -opioid receptor isoform polypeptide comprising steps of transfecting a recombinant host cell with a nucleic acid molecule consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 1 , 2 or a nucleic acid sequence having at least a 98% sequence homology to SEQ ID NO: 1 or 2 over its entire length or a nucleic acid sequence coding for a protein consisting essentially of an amino acid sequence of SEQ ID NO: 3 or for a protein comprising amino acids 1-300 of SEQ ID NO: 3, wherein the nucleic acid molecule encodes a six transmembrane ⁇ -opioid receptor isoform polypeptide; culturing the host cell under conditions sufficient for expression of the polypeptide; recovering the polypeptide; and preparing an antibody to the polypeptide.
  • a method of detecting a six transmembrane ⁇ -opioid receptor polypeptide comprising immunoreacting the polypeptide with an antibody prepared according to the method described above to form an antibody-polypeptide conjugate; and detecting the conjugate.
  • the presently disclosed subject matter futher includes a method of detecting a nucleic acid molecule that encodes a six transmembrane ⁇ -opioid receptor isoform polypeptide in a biological sample containing nucleic acid material, the method comprising the steps of hybridizing the nucleic acid molecule having a sequence complementary to one of SEQ ID NO: 1 or SEQ ID NO: 2 under stringent hybridization conditions to the nucleic acid material of the biological sample, thereby forming a hybridization duplex, and detecting the hybridization duplex.
  • an assay kit for detecting the presence of a six transmembrane ⁇ -opioid receptor isoform in a biological sample, the kit comprising a first container comprising a first antibody capable of immunoreacting with a polypeptide of any of the embodiments described above.
  • the kit further comprises a second container containing a second antibody that immunoreacts with the first antibody.
  • the first and second antibodies comprise monoclonal antibodies.
  • the first antibody is affixed to a solid support.
  • the first and second antibodies each comprise an indicator.
  • the indicator is a radioactive label or an enzyme.
  • an assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a six transmembrane ⁇ -opioid receptor isoform polypeptide, the kit comprising a polypeptide of any of the embodiments described above that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
  • an assay kit for detecting the presence, in biological samples, of a six transmembrane ⁇ -opioid receptor isoform polypeptide, the kit comprising a first container that contains a nucleic acid molecule identical or complementary to a segment of at least ten contiguous nucleotide bases of the nucleic acid molecule consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 1 , 2 or a nucleic acid sequence having at least a 98% sequence homology to SEQ ID NO: 1 or 2 over its entire length or a nucleic acid sequence coding for a protein consisting essentially of an amino acid sequence of SEQ ID NO: 3 or for a protein comprising amino acids 1-300 of SEQ ID NO: 3.
  • Figures 1 A-1 F are a series of images showing the expression pattern of exon 13 containing 0PRM1 gene splice variant in human and mouse.
  • Figure 1 A is an ethidium bromide gel analysis of RT-PCR performed on total RNA samples from the human brain regions known to express OPRM1 with hU2 (SEQ ID NO: 6) and hL5 (SEQ ID NO: 10) primers specific for exon
  • exon 13 and exon 2.
  • the exon 13 containing mu-opioid receptor (OPRM 1) gene splice variant MOR-1 K was detected in CNS but not in peripheral leukocytes even after a secondary PCR round with nested PCR primers.
  • the PCR product size was 1229 nucleotides (nt), which was 3 times longer than the predicted OPRM 1 gene splice variant MOR-1 K
  • Figure 1 B is an ethidium bromide gel analysis of RT-PCR analysis of mouse spinal cord with primer pairs mU2-mL3 (SEQ ID NOS: 18 and 21), and mU2-ml_1 (SEQ ID NOS: 18 and 20).
  • a longer mouse isoform orthologous to human exon 13 was below the level of detection even by secondary PCR with the nested PCR primers mU3-ml_3 (SEQ ID NOS: 19 and 21), mU3-mL1 (SEQ ID NOS: 19 and 20) or mU3-mL2.
  • Figure 1 C is a schematic diagram illustrating the relative position of PCR primers designed to amplify the new alternative MOR-1 K variant in mouse and human.
  • Figure 1 D is a schematic diagram illustrating the exonic composition and relative position of PCR primers designed to amplify the major MOR-1 variant and the newly identified alternative MOR-1 K variant.
  • the arrows indicate relative position of translation initiation start codons and stop codons.
  • Figure 1 E is a schematic illustration of the predicted protein structure of
  • MOR-1 and MOR-1 K isoforms. Translation of the MOR-1 K variants results in a 6TM domain receptor, truncated at the N-terminus.
  • Figure 1 F is an ethidium bromide gel analysis of RT-PCR results demonstrating the relative expression pattern of human MOR-1 (primers hU1- L3) and MOR-1 K (primers hU5-L3; SEQ ID NOS: 5 and 11) variants.
  • GAP3DH was used as a control for cDNA loading. All major PCR products resulting from this reaction were sequenced and aligned with human or mouse genomes.
  • Figure 2 is a bar graph showing the expression pattern of the MOR-1 K splice variant (primers hU2-L5) relative to canonical MOR-1 isoform (primers hU1-L5, SEQ ID NOS: 5 and 11) using real time PCR approach.
  • FIGS. 3A, 3B 1 and 4 are a set of graphs illustrating the cyclic adenosine monophosphate (cAMP)(Fig. 3A) and calcium (Ca 2+ )(FJg.
  • COS1 Fig. 3A 1 B
  • BE2C Fig.4 cells were transiently transfected with empty vector (Control), MOR1 (7TM) or MOR1 K (6TM) expressing constructs.
  • Figure 3A demonstrates that Forskoline (FSK, 10 ⁇ M) was used to increase cAMP levels prior to morphine treatment, which generally produced an inhibition of cAMP accumulation.
  • COS1 cells transfected with MOR1 showed decreased forskolin (FSK)-induced cAMP accumulation relative to cells transfected with empty vector following morphine treatment.
  • FSK forskolin
  • cells transfected with the MOR-1 K construct showed an increase in cAMP accumulation following morphine treatment, especially prominent in non-FSK- induced condition ( last bar).
  • Figure 3B is a bar graph showing that COS1 cells transfected with
  • MOR1 or empty vector did not exhibit morphine-induced changes in intracellular Ca ++ .
  • cells transfected with the MOR-1 K construct showed an increase in intracellular Ca ++ release following morphine treatment.
  • Figure 4 is a graph showing that Be2C cells transfected with MOR1 or empty vector did not exhibit morphine-induced changes in intracellular Ca ++ .
  • Data are Mean + SEM, * P ⁇ 0.05.Data are reported as mean ⁇ S.E.M from at least 6 experiments. * represent statistical significance different from the control vector condition. # represents a significant difference between the control vector condition and MOR1K transfected cells. All naloxone treatments were significantly different from the respective controls.
  • Tukey-Kramer method Data are presented as mean + SEM, # and *P ⁇ 0.05.
  • Figures 5A-5D are graphs showing nitric oxide (NO) production evoked by 7TM and 6TM mu-opioid receptor (OPRM 1) splice variants transiently transfected into cells.
  • BE2C cells were transiently transfected with MOR1K (6TM) or MOR1 (7TM) expressing constructs.
  • Figure 5A shows NO production in cells transfected with MOR1K in response to increasing concentrations of morphine or the OPRM1 antagonist naloxone (NaI).
  • the 6TM isoform (Fig. 5A), but not 7TM isoform (Fig.5B) produced robust concentration-dependent increases in the release of NO.
  • Figures 5C-D show that the time course response associated with morphine-induced NO release was markedly different when stimulating 6TM (Fig. 5C) and 7TM (Fig. 5D) isoforms. NO release was blocked by pretreatment with naloxone (0.1 ⁇ M) and was significantly different from the respective controls (not shown). ANOVA was used to determined main effects followed by post hoc testing using Tukey-Kramer method. Data are presented as mean + SEM, # and *P ⁇ 0.05.
  • Figure 6 is an immunoblotting of a set of pull-down assays.
  • the 6TM MOR-1 K Isoform couples to G ⁇ s (top left frame).
  • COS1 cells were transiently transfected with 7TM-MYC or 6TM-FLAG constructs.
  • the 6TM-FLAG construct (MOR1 K) was found to couple to G ⁇ s
  • the 7TM-MYC construct (MOR1) is coupled only to Gay 0 (two center bottom frames). Consistent with G ⁇ s binding to the 6TM isoform, morphine-mediated increases in Ca 2+ levels were not prevented by pre-treatment of the cells with pertussis (PTX) in 6TM-FLAG (far right frame).
  • Figure 7 is a set of graphs showing that mice that develop opioid- induced hyperalgesia exhibit increased spinal MOR-1 K transcript levels.
  • MOR-1 K plays a role in opioid-induced hyperalgesia by being upregulated following chronic opioid administration
  • C57BI/6J mice were treated with morphine (10-40 mg/kg) twice daily for 4 days and MOR-1 K RNA expression was measured at the time of peak hyperalgesia.
  • mice showed analgesia, such that (A) paw withdrawal frequency to a normally noxious punctuate mechanical stimulus and (B) the number of paw flicks and jumps on a 52.5°C hotplate were significantly decreased.
  • mice showed hyperalgesia, such that responses to mechanical and thermal stimuli were significantly increased.
  • mice were sacrificed and spinal cord tissue collected for measuring transcript levels using quantitative real-time PCR.
  • C Relative to vehicle-treated mice, morphine- treated mice did not exhibit changes in MOR1 RNA expression.
  • D They did, however, exhibit a significant 2-fold increase in MOR-1 K RNA expression. Data are Mean + SEM, *P ⁇ 0.05.
  • SEQ ID NO: 1 is a nucleotide sequence for homo sapiens ⁇ -opioid receptor-K1 (MOR-1 K1).
  • SEQ ID NO: 2 is a nucleotide sequence for homo sapiens ⁇ -opioid receptor-K2 (MOR-1 K2).
  • SEQ ID NO: 3 is an amino acid sequence for the six transmembrane (6TM) isoform of ⁇ -opioid receptor encoded by MOR-1 K1 and MOR-1 K2.
  • SEQ ID NO: 4 is an amino acid sequence for the seven transmembrane (7TM) isoform of ⁇ -opioid receptor (GENBANKTM Accession No. P35372).
  • SEQ ID NO: 5 is a nucleotide sequence for hU5, an upstream PCR primer for the human ⁇ -opioid receptor exon 13 mRNA.
  • SEQ ID NO: 6 is a nucleotide sequence for hU2, an upstream PCR primer for human ⁇ -opioid receptor exon 13 mRNA.
  • SEQ ID NO: 7 is a nucleotide sequence for hi 3U-1 , an upstream PCR primer for human ⁇ -opioid receptor exon 13 mRNA.
  • SEQ ID NO: 8 is a nucleotide sequence for h13U-2, an upstream PCR primer for human ⁇ -opioid receptor exon 13 mRNA.
  • SEQ ID NO: 9 is a nucleotide sequence for h13U-3, an upstream PCR primer for human ⁇ -opioid receptor exon 13 mRNA.
  • SEQ ID NO: 10 is a nucleotide sequence for hL5, a downstream PCR primer for human ⁇ -opioid receptor exon 2 mRNA.
  • SEQ ID NO: 11 is a nucleotide sequence for hL-3, a downstream PCR primer for human ⁇ -opioid receptor exon 4 mRNA.
  • SEQ ID NO: 12 is a nucleotide sequence for h5L-1 , a downstream PCR primer for human ⁇ -opioid receptor exon 5 mRNA.
  • SEQ ID NO: 13 is a nucleotide sequence for h5L-2, a downstream PCR primer for human ⁇ -opioid receptor exon 5 mRNA.
  • SEQ ID NO: 14 is a nucleotide sequence for h5L-3, a downstream PCR primer for human ⁇ -opioid receptor exon 5 mRNA.
  • SEQ ID NO: 15 is a nucleotide sequence for h5L-4, a downstream PCR primer for human ⁇ -opioid receptor exon 5 mRNA.
  • SEQ ID NO: 16 is a nucleotide sequence for hO/7L-1 , a downstream
  • SEQ ID NO: 17 is a nucleotide sequence for hO/7L-2, a downstream PCR primer for human ⁇ -opioid receptor exon 0/7 mRNA.
  • SEQ ID NO: 18 is a nucleotide sequence for mU2, an upstream PCR primer for murine ⁇ -opioid receptor exon 13 mRNA.
  • SEQ ID NO: 19 is a nucleotide sequence for mU3, an upstream PCR primer for murine ⁇ -opioid receptor exon 13 mRNA.
  • SEQ ID NO: 20 is a nucleotide sequence for ml_1 , a downstream PCR primer for murine ⁇ -opioid receptor exon 2 mRNA.
  • SEQ ID NO: 21 is a nucleotide sequence for ml_3, a downstream PCR primer for murine ⁇ -opioid receptor exon 2 mRNA.
  • SEQ ID NO: 22 is a nucleotide sequence for a PCR primer for MOR1.
  • SEQ ID NO: 23 is a nucleotide sequence for a PCR primer for MOR1.
  • SEQ ID NO: 24 is a nucleotide sequence for a PCR primer for MOR1 K.
  • SEQ ID NO: 25 is a nucleotide sequence for a PCR primer for MOR1 K.
  • SEQ ID NO: 26 is a nucleotide sequence for hOPRM1ex13-f, an upstream PCR primer for human OPRM1 exon 13.
  • SEQ ID NO: 27 is a nucleotide sequence for hOPRM1ex2-r, a downstream PCR primer for human OPRM1 exon 2.
  • SEQ ID NO: 28 is a nucleotide sequence for hOPRM1ex1-f, an upstream PCR primer for human OPRM1 exon 1.
  • SEQ ID NO: 29 is a nucleotide sequence for hOPRM1ex2-r(1), a downstream PCR primer for human OPRM1 exon 2.
  • the presently disclosed subject matter pertains to the existence of new exons within the human MOR-1. These exons contain several unexplored polymorphisms associated with individual variability in human pain sensitivity and responses to the MOR-1 agonist morphine. Importantly, these polymorphisms are situated within a newly identified MOR-1 isoform that encodes a truncated version of the classical MOR-1 (MOR-1 K), recently cloned as described herein. The presently disclosed subject matter relates to the newly discovered exon 13 within the human OPRM1 gene locus.
  • Exon 13- containing splice variants code for a six transmembrane (6TM) mu-opioid receptor-K (MOR-1 K) isoform that appears to have biological and clinical significance.
  • 6TM transmembrane
  • MOR-1 K mu-opioid receptor-K
  • MOR1 K is missing the extracellular and first transmembrane domain and, in contrast to the major MOR1 isoform, MOR1K activates a stimulatory Gas pathway and thereby elevates intracellular levels of cAMP, and Ca 2+ , and induces release of nitric oxide (NO).
  • NO nitric oxide
  • the newly identified polymorphisms are associated with pain and morphine responses, their activation leads to excitatory signaling, and their RNA levels are increased in mice with opioid hyperalgesia. Therefore, the major and the alternative MOR variants mediate opposite cellular effects and thus the balance between these isoforms can influence therapeutic outcome of opioid treatment.
  • 6TM Six-Transmembrane
  • MOR-1K Human Mu-Opioid Receptor
  • the human exon 13 containing isoform analogous to mouse MOR-1 K isoform, appears to be of particular interest due to relative position of the functional SNP rs563649 (Figure 1C) situated within the exon 13 conservation area between mouse and human genomes (see WO 07/070252, incorporated herein by reference in its entirety).
  • SNP rs563649 which is significantly associated with pain sensitivity and morphine analgesic efficacy, is located within a structurally conserved internal ribosomal binding site (IRES) in the 5'UTR of this novel exon 13-containing isoform and affects MOR-1 K translation efficiency.
  • IRS internal ribosomal binding site
  • MOR-1 K RNA is enriched in spinal cord and brain.
  • the presently disclosed subject matter demonstrates that stimulation of MOR-1 K in vitro leads to cellular excitation characterized by increased cAMP, Ca 2+ , and NO production and that MOR-1 K RNA expression is increased in mice exhibiting opioid-induced hyperalgesia.
  • the human MOR-1 K isoform codes for a novel human truncated version of OPRM1 that includes 6, rather than 7, trans-membrane (TM) domains and lacks an extracellular domain.
  • TM trans-membrane
  • the extracellular N-terminus and first cytoplasmic domain are missing in this isoform. Instead, it possesses a cytoplasmic N-terminus followed by 6 transmembrane domains and C-terminus that do not differ from the 7TM isoform.
  • This truncated 6TM receptor should retain a ligand binding pocket that is distributed across the conserved TMH2, TMH3, and TMH7 domains of MOR1 (Edwards et al., (2006) Anesthesiology, 104, 1243-1248), which means that the 6TM isoform should be capable of binding MOR agonists.
  • MOR-1 K is an example of a functional 6TM isoform.
  • allelic variants coding for higher MOR1K expression was associated with greater sensitivity to noxious stimuli and poorer responses to morphine (Sora 1997), which is opposite from what one would expect in response to MOR activition.
  • 6TM MOR receptor isoform activity contributes to hyperalgesic effects of MOR agonists throughout stimulation cellular excitatory pathways.
  • MOR-1 K codes for a truncated 6TM OPRM1 receptor with a different intracellular domain and tissue-specific distribution as compared to a MOR-3 variant reported previously (see Cadet et al., J. Immunol., 170, 5118- 5123 (2003). In further contrast to MOR-3, MOR-1 K expresses in brain tissues and neuronal cells but not in vascular tissues or leukocytes.
  • RT-PCR was performed using RNA isolated from the human brain tissues known to respond to opioid treatment and that express high levels of OPRM 1 (Figure 2).
  • the forward primers were designed to predicted exon 13 and reverse primers to predicted exon 2.
  • Figures 1A-1C RT-PCR results show that the human exon 13 is approximately 0.8 kb longer than the mouse exon 13 and carries alternative acceptor sites of splicing similar to OPRM1 exons 1 , 3 and 5. See Pasternak, Neuropharmacology, 47 Suppl 1, 312-323 (2004); and Pan, DNA Cell Biol., 24, 736-750 (2005).
  • the RT-PCR results reveal MOR-1 K expression in all examined brain tissues that are known to express MOR1 and which contribute to the pharmacological effects of MOR agonists, (Fig.2), including frontal lobe, medulla oblongata, insula, nucleus accumbens, pons, spinal cord, dorsal root ganglion (DRG). Amplification of MOR-1 K isoforms from connective tissues surrounding DRG and peripheral leukocytes was not seen. Furthermore, the human neuroblastoma cell lines Be2C and SY5Y showed the highest expression of MOR-1 K isoforms while monkey kidney COS-1 and human astrocytoma H4 cell lines did not express M0R-1K. Together, these data indicate a restricted expression of M0R-1K isoforms in neuronal cells with its highest relative expression in transformed neuronal cell lines suggesting a suppression of MOR- 1 K expression in native cellular conditions.
  • the sequencing results of the RT-PCR product amplified from frontal lobe, nucleus accumbens, medulla oblongata and spinal cord identified a 5 1 UTR and a partial coding region of novel OPRM1 splicing isoforms MOR-1 K1 and MOR-1 K2, which are divergent in their 3'exon boundaries sites by 12 nucleotides (nt).
  • Both human MOR-1 K1 and MOR-1 K2 isoforms encode a truncated version of OPRM1 that lacks an extracellular N-terminal domain and transmembrane domain I, and, thus, consists of 6 transmembrane domains (6TM) instead of the classic 7TM characteristic of major OPRM 1 isoforms.
  • 6TM transmembrane domains
  • This truncated 6TM receptor is believed to retain a ligand binding pocket that is distributed across the conserved TMH2, TMH3, and TMH7domains of OPRM1 (see Fowler et al., Biochem., 43, 15796-15810 (2004)), which means that 6TM isoforms should be capable of binding OPRM1 agonists.
  • Expression vectors with cloned coding regions of 7TM (MOR-1) or 6TM (MOR-1 K) OPRM1 receptor variants were transfected into immortalized cell lines in order to characterize the cellular effects of the human 6TM isoform on cAMP and Ca 2+ response.
  • the transfected cell lines included a cell line that expresses endogenous OPRM1 (i.e., the BE2C human neuroblastoma cell line) and a cell line that does not express endogenous OPRM1 (i.e., African green monkey kidney COS1 cell line).
  • VGCC voltage-gated Ca 2+ channels
  • Polomano et a ⁇ .,Semin. Perioper. Nurs. 10, 3-16 (2001); Polomano et al., Semin. Perioper. Nurs., 10, 159-166 (2001) the cellular characterization of cAMP accumulation and intracellular Ca 2+ levels was used to assess the functional effects of MOR1 activation (Polomano et al., Semin. Perioper.
  • COS1 cells transfected with the 6TM isoform do not show a morphine- dependent decrease in forskolin induced cAMP levels, but instead show a very substantial increase in cAMP levels that is especially apparent in the forskolin- free condition (Fig. 3A).
  • COS1 cells transfected with MOR1K construct showed a substantial increase in intracellular Ca 2+ release following morphine treatment (Fig. 3B), while MOR1 isoform does not show morphine- induced changes in unstimulated cells with basal Ca 2+ levels. Similar results were obtained in Be2C neuroblastoma cells.
  • VGCC appears to be the primary target underlying the rapid inhibitory effects of opioids (Chou et al., Acta Anaesthesiol. Scand, 50, 787- 792 (2006); Skarke et al., CHn. Pharmacol. Ther, 73, 107-121 (2003)), and morphine stimulation of the 6TM isoform seems to increase intracellular Ca 2+ levels (Fig. 3B), the dose-dependent regulation of Ca 2+ levels in BE2C cells transiently transfected with 6TM (Fig. 4) was examined.
  • BE2C cells showed a robust dose-dependent morphine-induced increase in Ca 2+ levels in cells transfected with the 6TM isoform; a response that was blocked by the opioid receptor antagonism with naloxone.
  • the increase in Ca 2+ levels was significantly different from the increases observed in BE2Cs transiently transfected with either the 7TM isoform or empty vector.
  • COS1 cells transfected with the major 7TM form of OPRM1 showed decreased forskolin- induced (FSK; 10 ⁇ M) cAMP accumulation relative to non-transfected cells in response to treatment with morphine.
  • FSK forskolin- induced
  • COS1 cells transfected with 6TM construct showed an increase in FSK evoked cAMP accumulation. See Figure 3A.
  • Treatment of unstimulated COS1 cells transfected with the major 7TM form of OPRM1 or an empty vector with 1 ⁇ M morphine did not change the amount of intracellular Ca ++ (not shown).
  • COS1 cells transfected with the 6TM form of OPRM 1 showed an increased release of intracellular Ca ++ after treatment with 1 ⁇ M of morphine.
  • Figure 3B Very similar results were obtained using the BE2C cell line.
  • Morphine-dependent nitric oxide (NO) production was also characterized in neuroblastoma Be2C cells. NO production is a cellular process that is believed to contribute to morphine induced hyperalgia. See Mayer, et al., Proc, Natl., Acad, ScL U.S.A., 96, 7731-7736 (1999).
  • 6TM transfected Be2C cells showed robust NO production in response to increasing concentrations of morphine. There were marked differences between 7TM and 6TM MORs with respect to morphine-induced NO-production in the transfected Be2C cells.
  • Activation of 6TM transfected cells were far more sensitive to morphine resulting in a substantial dose-dependent increase in NO in 6TM transfected cells, with morphine concentrations 2-3 orders of magnitude lower than that observed with 7TM isoform stimulation (Figs.5A-5D).
  • Pretreatment with the specific OPRM 1 antagonist naloxone attenuated the increase in production of NO (Fig. 5A), resulting in a rightward shift of the dose response curve.
  • Be2C cells transfected with major 7TM OPRM1 form also showed a significant rightward shift of the dose response curve in comparison to cells transfected with 6TM OPRM1 form (see Figure 5B).
  • GPCR signaling results from a dissociation of the heterotrimeric G- protein complex.
  • Uncoupling of G ⁇ -G ⁇ 0 causes an inhibition of adenylate cyclase (AC) resulting in a decrease in cAMP production and the release of ⁇ y subunits which inhibits Ca 2+ channels leading to an inhibition of neural activity.
  • AC adenylate cyclase
  • ⁇ y subunits which inhibits Ca 2+ channels leading to an inhibition of neural activity.
  • uncoupling of Gas and G ⁇ q subunits results in increase levels of cAMP followed by cellular excitation.
  • Electrophysiological studies of the effects of opioids on nociceptive-like dorsal root ganglion (DRG) neurons in culture have provided in vivo evidence that the inhibitory effects (e.g.
  • MOR-1 K isoform leads to cellular excitation characterized by increased levels of cAMP and Ca 2+ and increased NO production.
  • MOR-1 K codes for a truncated 6TM OPRM 1 receptor with a different intracellular domain and tissue-specific distribution as compared to to the MOR-3 variant reported previously (see Cadet et al., J. Immunol., 170, 5118-5123 (2003)).
  • MOR-1 K expresses in brain tissues and neuronal cells but not in vascular tissues or leukocytes, suggesting higher relevance to morphine analgesia.
  • MOR-1 K lacks the unique intracellular C-terminal amino acid sequences that are characteristic for MOR-3 and which have been hypothesized to serve as coupling or docking domains required for constitutive NO synthase (NOS) activation (see Kream etal., Med. ScL Monit, 13, SC5-SC6 (2007)), MOR-1 K stimulation leads to NO production similar to MOR-3.
  • NOS constitutive NO synthase
  • the results have very broad basic cell molecular and medical implications. First, they significantly contribute to the understanding of the molecular and cellular biology of MOR receptor variants and GPCRs.
  • the MOR1 K isoform is a MOR alternatively spliced form coding for a truncated version of MOR that lacks an extracellular N-terminal domain and transmembrane domain I resulting in a 6TM rather than the classic 7TM receptor variant.
  • the results from the presently disclosed subject matter are the first to show that 6TM receptor activation results in increases in the intracellular production of excitatory mediators (cAMP, Ca 2+ and NO), while activation of 7TM results in the inhibition of cellular activity.
  • cAMP excitatory mediators
  • opioids such as morphine.
  • the up-regulation of this isoform would lead to changing balance between 7TM and 6M relative activities and overall analgesic outcome of the MOR agonists treatment.
  • MOR agonists are amongst most widely used analgesics, prescribed for both acute postoperative pain and chronic pain conditions; yet, there are substantial drug induced side effects for which there is very limited understanding. Thus, the further understanding of the molecular and cellular mechanisms that contribute to the analgesic, hyperalgesic, and analgesic tolerance effects of opioids is needed.
  • the present results provide substantial evidence that the 6TM MOR isoform is not just another alternatively-spliced form of MOR1 , but instead contributes to the net therapeutic effects of MOR agonists by facilitating the excitatory responses to opioids and potentially represents a molecular target that mediates OIH, and analgesic tolerance.
  • 6TM and 7TM MOR receptor variants The elucidation of the biological and cellular properties engaged by 6TM and 7TM MOR receptor variants, can ultimately lead to the identification and development of a new class of opioid analgesics that show a high degree of analgesic efficacy with fewer side effects.
  • both the agonist and antagonist effects on 6TM and 7TM MOR isoforms can be considered.
  • the presently claimed subject matter includes a method for identifying an antagonist or an agonist specific for a 6TM ⁇ -opioid receptor isoform.
  • the isoform comprises a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology thereto over the entire length of the amino acid.
  • the method comprises contacting a cell with a test molecule and a ⁇ -opioid receptor agonist or antagonist, wherein said cell comprises a nucleic acid molecule consisting essentially of one of SEQ ID NOs: 1 or 2 or a sequence having at least a 98% homology thereto, and wherein said cell expresses said polypeptide; and determining whether or not said test molecule reduces or prevents a non- analgesic or hyperanalgesic ⁇ -opioid receptor-mediated response induced by said ⁇ -opioid receptor agonist or antagonist.
  • MOR1 K and MOR1 expression constructs were subcloned into plRES2- EGFP (Clontech Laboratories, Inc., Mountain View, California, United States of America) expression vector using Sacl/Sacll restriction sites.
  • MOR PCR products were generated using following primers: for MOR1 TATCGAGCTCGCCACCATG-GACAGCAGCGCTGCCCCCACGAAC (SEQ ID NO: 22) and ATCCCCGCGGTT-AGGGCAACGGAGCAGTTTCTGCTTC (SEQ ID NO: 23); for MOR1K
  • BE2C and COS1 cells were obtained from ATCC.
  • the cells we grown to 90% confluence and transfected with MOR1K, MOR1 or GFP expression constructs with Lipofectamin 2000 reagent using manufacturer guidelines.
  • Opticon-2 Real Time Fluorescence Detection System (MJ Research) was used for measuring fluorescence.
  • BE2C human neuroblastoma cells (American Type Culture Collection - ATCC) were grown to near confluence in black 96-well poly-D-lysine coated plates. The cell cultures were grown in DMEM/F12 media.
  • the indicator Fluo-4 NW dye (Invitrogen) was prepared as outlined in manufacturer instructions. 10O ⁇ L of Fluo-4 NW dye was added to each well. The plate was then incubated with the lid on at 37°C for 30 minutes, then at room temperature for an additional 30 minutes. The fluorescence was measured using Victror-3 (Perkin Elmer, Waltham, Massachusetts, United States of America) microplate reader with settings for emission at 515 nm and excitation at 500 nm.
  • Nitric oxide (NO) measurement Cells were grown in DMEM/F12 supplemented with 10% FBS at 37°C.
  • NO release from the transfected and untransfected cell lines is directly measured using an NO-specific amperometric probe.
  • the amperometric probe was allowed to equilibrate for at least 10 min before being transferred to the well containing the cells.
  • Morphine-stimulated NO release was evaluated in response to increasing morphine concentrations, such as in the range of 10 "5 — 10 "9 M. Each experiment is repeated four times along with a control (cells transfected with vector alone). CyAMP assay
  • MOR or MOR1 K expressants were plated on 12 well plates and grown to 90% confluency. On the day of sample preparation, cells were washed with DMEM to remove serum and incubated with serum-free DMEM containing the phosphodiesterase inhibitor 100 ⁇ M of IBMX (Sigma-Aldrich, St. Louis, Missouri, United States of America) for 30 min, morphine was then added and cells incubated for a further 15 min. Following this, forskoline FSK (50 ⁇ M) was added to the wells and the cells were incubated for 15 min to stimulate cAMP production. DMSO alone was used as a vehicle control.
  • IBMX Sigma-Aldrich, St. Louis, Missouri, United States of America
  • the cells were lysed using RIPA buffer and centrifuged at maximum RPM for 20 minutes. Supernatants were collected and used for Co-Ip experiments. Following the overnight incubation on the rotary shaker at 4°C overnight with anti-FLAG (MOR1 K) or anti-MYC (MOR1 ) antibodies the beads (Pierce Biotechnology, Rockford, Illinois, United States of America) were added and the samples were incubated for 6 hours at 4°C. The samples were then centrifuged at 14000 rpm for 10 min, the supernatants were discarded and the beads were re-suspended in 50% RIPA/PBS buffer. The procedure was repeated 3 times.
  • MOR1 K anti-FLAG
  • MOR1 anti-MYC
  • the blots were incubated for 4 hours at room temperature with a horseradish peroxidase-conjugated secondary antibody (1 :15,000; GE Healthcare Bio-Sciences). All antibodies were diluted in blocking buffer. The antibody-antigen complexes were detected using the ECL system (Amersham, Pittsburgh, Pennsylvania, United States of America) and visualized with photosensitive film (Kodak, Rochester, New York, United States of America).
  • FIG. 7 exhibits representative data showing that in C57BI/6J mice morphine suppresses both noxious mechanical and thermal pain perception and that repeated daily administration of escalading doses of morphine over a 4 day period results in marked signs of mechanical (Panel A) and thermal (Panel B) hyperalgesia.
  • OIH opioid induced hyperalgesia
  • mice Sixteen male and sixteen female adult C57B/6J, CXBK, and 129 SV/EV mice (20-3Og; Jackson Labs, Bar Harbor, Maine, United States of America, and Taconic, Germantown, New York, United States of America) were used in these experiments. All procedures were approved by the University of North Carolina Animal Care and Use Committee and adhered to the guidelines of the Committee for Research and Ethical Issues of the IASP (Zimmermann, 1993).
  • Drugs and Chemicals Morphine sulfate was dissolved in 0.9% saline with volumes depending on the desired ending doses of 10 mg/kg, 20 mg/kg, or 40 mg/kg.
  • the injection volumes were 20-30 uL depending on the weight of the animal.
  • the morphine sulfate was obtained from the National Institute for Drug Abuse.
  • mice were handled and habituated to the testing environments for 4 days prior to establishing baseline responsiveness to all tests. On testing days, mice were placed in the open field infrared tracking system and their movements were tracked for a 15 minute period. The number of feces pellets produced by each mouse was noted at the conclusion of the 15 minute period.
  • mice were then placed in PlexiglasTM cages positioned over an elevated perforated stainless steel platform and habituated to the environment for 10 minutes priorto the mechanical testing. Afterthe conclusion of the mechanical testing, mice were individually placed onto a hot plate and their reactions were videotaped for a period of one minute. Mice were then returned to their home cages.
  • mice were baselined for one day. The mice then received either morphine or vehicle subcutaneous injections twice daily for 4 days. On day 1 , the morphine dosage was 10 mg/kg. Days 2 and 3 had a morphine dosage of 20 mg/kg and the final day had a morphine dosage of 40 mg/kg.
  • the behavior testing regimen was conducted once per group in the morning. During days the mice received injections, mice were tested both before and following the injection in the morning. The mice were also tested in the open field infrared tracking device prior to and following the night injection.
  • mice where placed into the PlexiglasTM container where they had free access to move around. Their movements were tracked for a 15 minute period and then analyzed for total distance traveled. After the mice were removed from the container, the number of feces pellets was recorded in order to determine defecation rates.
  • Paw withdrawal threshold was assessed using the up-down method (Chaplan et al., J Neurosci Methods 53:55-63 (1994)) to determine the threshold for punctate mechanical stimulation.
  • a series of eight calibrated filaments (with bending forces of 0.07, 0.17, 0.41 , 0.70, 1.19, 1.50, 2.05, 3.63g; (Stoelting Co., Wood Dale, Illinois, United States of America)) were presented to the hind paw in successive order, whether ascending or descending. Filaments were positioned in contact with the hind paw for a duration of 3 s or until a withdrawal response occurred. Testing was initiated with the middle hair of the series (0.70 g).
  • paw withdrawal frequency (%) to punctate mechanical stimulation was assessed.
  • a von Frey monofilament with a calibrated bending force of 0.40 g was presented to the hind paw ten times for a duration of 1 s with an interstimulus interval of approximately 1 s.
  • Mechanical allodynia was defined as an increase in the percentage frequency ([No. of paw withdrawals/10] 100) of paw withdrawal evoked by stimulation with von Frey monofilaments. After the all of the animals were tested with the 0.40 g monofilament, the process was repeated with a 1.50 g force monofilament to determine mechanical hyperalgesia.
  • Thermal hyperalgesia was evaluated using the hot plate method. Mice were placed in a hot plate behavior testing apparatus (Columbus Instruments, Columbus, Ohio, United States of America) with a plate temperature of 51.5°C for a period of one minute. Mice were videotaped for this duration and then later analyzed for behavioral actions. The number of jumps and paw flicks (any paw) were recorded for each mouse during the testing period. The number of these actions where combined to create a "total reactions" score for each animal.
  • nucleic acid and “nucleic acid sequence” as used herein encompass both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
  • an isolated nucleic acid of amino acid sequence is one that is substantially free of the materials with which it is associated in its native environment. In some embodiments, it is meant at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% free of such materials.
  • the term "isolated" as used herein with reference to nucleic acid sequences refers to a naturally-occurring nucleic acid sequence that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3 1 end) in the naturally-occurring genome of the organism from which it is derived.
  • an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally- occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.
  • an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.
  • isolated as used herein with reference to nucleic acids also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
  • non-naturally-occurring nucleic acids such as an engineered nucleic acid can be considered to be isolated nucleic acid.
  • Engineered nucleic acids can be made using common molecular cloning or chemical nucleic acid synthesis techniques.
  • Isolated non-naturally-occurring nucleic acids can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote.
  • a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.
  • exogenous as used herein with reference to nucleic acids ( or with reference to amino acid sequences or peptides) and a particular cell refers to any nucleic acid that does not originate from that particular cell as found in nature. Thus, all non-naturally-occurring nucleic acids are considered to be exogenous to a cell once introduced into the cell. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid sequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature.
  • a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a cell once introduced into the cell, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature.
  • any vector, autonomously replicating plasmid, or virus e.g., retrovirus, adenovirus, or herpes virus
  • genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acids since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally- occurring nucleic acid.
  • a nucleic acid that is naturally-occurring can be exogenous to a particular cell.
  • an entire chromosome isolated from a cell of animal X is an exogenous nucleic acid with respect to a cell of animal Y once that chromosome is introduced into Y's cell.
  • nucleic acid sequences consisting essentially of a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the presently disclosed nucleic acid sequences relate to exon 13-containing splice variants of the human OPRM1 gene.
  • the nucleic acid sequences comprise exons 13, 2, 3, and 4.
  • consisting essentially of is meant a sequence that is at least 95%, 96%, 97%, 98%, or 99% homologous to a given sequence, such as SEQ ID NO: 1 or SEQ ID NO: 2.
  • the sequence will be at least 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 1 or SEQ ID NO: 2 over the entire length of the sequence.
  • the sequence that consists essentially of SEQ ID NO: 1 or SEQ ID NO: 2 includes one nucleotide addition, deletion or substitution.
  • the nucleic acid consists of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the presently disclosed subject matter provides an isolated nucleic acid sequence that codes for a protein consisting essentially of SEQ ID NO: 3.
  • the sequence encodes for a protein comprising amino acids 1-300 of SEQ ID NO: 3 or that consists essentially of amino acids 1-300 over the entire length of the amino acid sequence.
  • the isolated nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a contiguous sequence of the coding region of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the isolated nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a contiguous 902 nucleotide sequence selected from nucleotides
  • Sequence identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group.
  • the GAP program utilizes the alignment method of Needleman et al. (1970) J MoI Biol 48:443, as revised by Smith et al. (1981) Adv Appl Math 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. Representative parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See Schwartz et al.
  • the presently disclosed subject matter provides a nucleic acid that is complementary to the MOR-1 K splice variant (e.g., MOR- 1K1 or MOR-1 K2).
  • Nucleic acid sequences which are "complementary” are those, which are base-paired according to the standard Watson-Crick complementarity rules.
  • the term "complementary sequences” means nucleic acid sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein.
  • a particular example of a provided complementary nucleic acid segment is an antisense oligonucleotide.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of about 30 0 C, typically in excess of about 37°C, and optionally in excess of about 45°C.
  • Stringent salt conditions will ordinarily be less than about 1 ,000 mM, typically less than about 500 mM, and optionally less than about 200 mM.
  • stringent hybridization scheme uses 50% formamide, 4XSSC at 42°C.
  • stringent conditions means conditions of high stringency, for example 6XSSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 ⁇ g/ml salmon sperm DNA and 15% formamide at 68°C.
  • Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M NaCI/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC. Sequence identity can be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM NaCI/0.9 mM sodium citrate).
  • Vectors The presently disclosed subject matter further encompasses MOR-1 K splice variant polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element if necessary.
  • a “vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo.
  • the heterologous polynucleotide can comprise a sequence of interest for purposes of therapy or biomedical or genetic research, and can optionally be in the form of an expression cassette.
  • a vector need not be capable of replication in the ultimate target cell or subject.
  • the term includes cloning vectors for translation of a polynucleotide encoding sequence. Also included are viral vectors.
  • recombinant means a polynucleotide of genomic cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.
  • Heterologous means derived from a genetically distinct entity from the rest of the entity to which it is being compared.
  • a polynucleotide can be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
  • polynucleotides of the presently disclosed subject matter can comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct desirable to provide embodiments of the presently disclosed subject matter.
  • a "host cell” denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector.
  • the term refers both to the originally altered cell and to the progeny thereof.
  • Polynucleotides comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification.
  • Polynucleotides can be introduced into host cells by any means known in the art.
  • the vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including direct uptake, endocytosis, transfection, f-mating, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is infectious, for instance, a retroviral vector).
  • the choice of introducing vectors or polynucleotides will often depend on features of the host cell.
  • the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome.
  • Amplified DNA can be isolated from the host cell by standard methods. See, e.g., Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, incorporated herein in its entirety.
  • RNA can also be obtained from transformed host cell, or it can be obtained directly from the DNA by using a DNA-dependent RNA polymerase.
  • polyadenylation site e.g., 5 ' -AATAAA-3 '
  • these poly-A addition sites are placed about 30 to 2000 nucleotides "downstream" of the coding sequence at a position prior to transcription termination.
  • control sequences of the specific gene While use of the control sequences of the specific gene will be preferred, other control sequences can be employed, so long as they are compatible with the genotype of the cell being treated.
  • useful promoters including, e.g., an SV40 early promoter, a long terminal repeat promoter from retrovirus, an actin promoter, a heat shock promoter, a metallothionein promoter, and the like.
  • a promoter is a region of a DNA molecule typically within about 100 nucleotide pairs upstream of (i.e., 5' to) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes.
  • an enhancer imposes specificity of time, location and expression level on a particular coding region or gene.
  • a major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer.
  • An enhancer can function when located at variable distances from transcription start sites so long as a promoter is present.
  • the phrase "enhancer-promoter” means a composite unit that contains both enhancer and promoter elements.
  • An enhancer- promoter is operatively linked to a coding sequence that encodes at least one gene product.
  • the phrase "operatively linked” means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter.
  • Approaches for operatively linking an enhancer-promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the enhancer-promoter.
  • An enhancer-promoter used in a vector construct of the presently disclosed subject matter can be any enhancer-promoter that drives expression in a cell to be transfected.
  • an enhancer-promoter with well- known properties, the level and pattern of gene product expression can be optimized.
  • Viral vectors can be used. These vectors can be an adenoviral, a retroviral, a vaccinia viral vector, adeno-associated virus or Lentivirus; these vectors have been successfully used to deliver desired sequences to cells and tend to have a high infection efficiency. Suitable vector- target gene constructs are adapted for administration as pharmaceutical compositions, as described herein below. Viral promoters can also be of use in vectors of the presently disclosed subject matter, and are known in the art.
  • viral promoters for expression vectors are derived from polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40).
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment that also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided there is included the approximately 250 base pair sequence extending from the Hind III site toward the BgI I site located in the viral origin of replication.
  • promoter or control sequences normally associated with the desired gene sequence provided such control sequences are compatible with the host cell systems.
  • the origin of replication can be provided either by construction of the vector to include an exogenous origin, such as can be derived from SV40 or other viral source, or can be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • an expression vector comprising a polynucleotide that encodes a biologically active mu-opioid receptor isoform polypeptide in accordance with the presently disclosed subject matter.
  • an expression vector of the presently disclosed subject matter comprises a polynucleotide that encodes a polypeptide consisting essentially of SEQ ID NO: 3 or of amino acids 1-300 of SEQ ID NO: 3.
  • an expression vector of the presently disclosed subject matter comprises a polynucleotide comprising the nucleotide sequence consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 2.
  • an expression vector of the presently disclosed subject matter comprises a polynucleotide operatively linked to an enhancer-promoter.
  • an expression vector of the presently disclosed subject matter comprises a polynucleotide operatively linked to a prokaryotic promoter.
  • an expression vector of the presently disclosed subject matter comprises a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter and the expression vector further comprises a polyadenylation signal that is positioned 3' of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide.
  • the presently disclosed subject matter provides a recombinant host cell transfected with a polynucleotide that encodes a biologically active mu-opioid receptor isoform polypeptide in accordance with the presently disclosed subject matter.
  • a recombinant host cell of the presently disclosed subject matter is transfected with the polynucleotide that encodes human MOR-IK polypeptide.
  • a recombinant host cell of the presently disclosed subject matter is transfected with the polynucleotide sequence encoding or set forth in any of SEQ ID NOs: 1-2.
  • a recombinant host cell is a mammalian cell.
  • a recombinant host cell of the presently disclosed subject matter is a prokaryotic host cell, including parasitic and bacterial cells.
  • a recombinant host cell of the presently disclosed subject matter is a bacterial cell, such as but not limited to a strain of Escherichia coli.
  • a recombinant host cell can comprise a polynucleotide under the transcriptional control of regulatory signals functional in the recombinant host cell, wherein the regulatory signals appropriately control expression of the mu- opioid receptor polypeptide in a manner to enable all necessary transcriptional and post-transcriptional modification.
  • substantially pure polypeptides The term "substantially pure” as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
  • any suitable method can be used to obtain a substantially pure polypeptide.
  • common polypeptide purification techniques such as affinity chromotography and HPLC as well as polypeptide synthesis techniques can be used.
  • any material can be used as a source to obtain a substantially pure polypeptide.
  • tissue from wild-type or transgenic animals can be used as a source material.
  • tissue culture cells engineered to over-express a particular polypeptide of interest can be used to obtain substantially pure polypeptide.
  • a polypeptide within the scope of the presently disclosed subject matter can be engineered to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix.
  • a tag such as c-myc, hemagglutinin, polyhistidine, or FLAGTM tag (Kodak) can be used to aid polypeptide purification.
  • tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini.
  • Other fusions that could be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.
  • consisting essentially of refers to an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence homology to a give sequence, such as SEQ ID NO: 3.
  • the amino acid comprises a sequence consisting of amino acids 1-300 of SEQ ID NO: 3.
  • the amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% sequence homology to amino acids 1- 300 of SEQ ID NO: 3 over the entire length of the sequence.
  • the polypeptide consists of SEQ ID NO: 3.
  • the terms "6TM mu-opioid receptor” and “6TM mu-opioid receptor isoform” refer to a polypeptide consisting essentially of SEQ ID NO: 3 or encoded by the nucleic acid sequence consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 2.
  • “7TM mu-opioid receptor” or “7TM mu-opioid receptor isoform” refer to a polypeptide consisting essentially of SEQ ID NO: 4 or comprising the sequence consisting essentially of amino acids 67- 330 of SEQ ID NO: 4.
  • modifications and changes can be made in the structure of the mu-opioid receptor isoform proteins and peptides described herein and still constitute a molecule having like or otherwise desirable characteristics.
  • certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive capacity with, for example, structures in the nucleus of a cell. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or the nucleic acid sequence encoding it) to obtain a protein with the same, enhanced, or antagonistic properties.
  • Biologically functional equivalent peptides are peptides in which certain, but not most or all, of the amino acids can be substituted.
  • amino acids can be substituted.
  • applicants provide substitution of codons that encode biologically equivalent amino acids as described herein into the sequence examples of SEQ ID NO: 3.
  • amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience.
  • functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man can be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine a desired activity at the molecular level.
  • Amino acid substitutions such as those which might be employed in modifying the mu-opioid receptor isoform proteins and peptides described herein, are generally based on the relative similarity of the amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.
  • arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are defined herein as biologically functional equivalents.
  • Other biologically functionally equivalent changes will be appreciated by those of skill in the art.
  • hydropathic index of amino acids can be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (- 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartate (+ 3.0 ⁇ 1); glutamate (+ 3.0 ⁇ 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-
  • Recombinant vectors and isolated DNA segments can therefore variously include the mu-opioid receptor polypeptide-encoding region itself, include coding regions bearing selected alterations or modifications in the basic coding region, or include larger polypeptides which nevertheless comprise mu-opioid receptor polypeptide-encoding regions or can encode biologically functional equivalent proteins or peptides which have variant amino acid sequences.
  • the presently disclosed subject matter further encompasses fusion proteins and peptides wherein the mu-opioid receptor isoform coding region is aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes.
  • peptidyl compounds described herein can be formulated to mimic the key portions of the peptide structure.
  • Such compounds can be used in the same manner as the peptides of the presently disclosed subject matter and hence are also functional equivalents.
  • the generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the presently disclosed subject matter.
  • transgenic non-human animals that express a human mu- opioid receptor splice variant (e.g., MOR-1K1 or MOR-1K2) or that have modified OPRM1 expression.
  • a representative transgenic animal is a mouse.
  • transgenic rats transgenic rats
  • U.S. Patent Nos.4,736,866, 5,550,316, 5,614,396, 5,625,125 and 5,648,061 transgenic mice
  • U.S. Patent No. 5,573,933 transgenic pigs
  • 5,162,215 transgenic avian species
  • U.S. Patent No. 5,741,957 transgenic bovine species
  • transgenic animals with a specific "knock-out" modification.
  • the transgenic animal can be provided that under expresses a seven-transmembrane (7TM) mu-opioid receptor isoform.
  • a knockout of a target gene means that function of the gene has been substantially decreased so that expression is not detectable or only present at insignificant levels. This can be achieved by a variety of mechanisms, including introduction of a disruption of the coding sequence, e.g. insertion of one or more stop codons, insertion of a DNA fragment, etc., deletion of coding sequence, substitution of stop codons for coding sequence, etc.
  • a chromosomal deletion of all or part of the native gene can be induced, including deletions of the non-coding regions, particularly the promoter region, 3 1 regulatory sequences, enhancers, or deletions of gene that activate expression of target genes.
  • Afunctional knock-out can also be achieved by the introduction of an anti-sense construct that blocks expression of the native genes (for example, see Li and Cohen (1996) Ce// 85:319-329).
  • "Knockouts” also include conditional knock-outs, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
  • the presently disclosed subject matter provides an antibody immunoreactive with a polypeptide of the presently disclosed subject matter.
  • an antibody of the presently disclosed subject matter is a monoclonal antibody.
  • Techniques for preparing and characterizing antibodies are well known in the art (See e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide or polynucleotide of the presently disclosed subject matter, and collecting antisera from that immunized animal.
  • a wide range of animal species can be used for the production of antisera.
  • a rabbit is a representative choice for production of polyclonal antibodies.
  • a given polypeptide or polynucleotide can vary in its immunogenicity. It is often necessary therefore to couple the immunogen (e.g., a polypeptide or polynucleotide) of the presently disclosed subject matter) with a carrier.
  • exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • immunogenicity to a particular immunogen can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants.
  • adjuvants include complete
  • the amount of immunogen used of the production of polyclonal antibodies varies, inter alia, upon the nature of the immunogen as well as the animal used for immunization.
  • routes can be used to administer the immunogen, e.g., subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal.
  • the production of polyclonal antibodies is monitored by sampling blood of the immunized animal at various points following immunization. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored.
  • the presently disclosed subject matter provides a process of producing an antibody immunoreactive with a six transmembrane mu-opioid receptor isoform polypeptide, the process comprising the steps of (a) transfecting recombinant host cells with a polynucleotide that encodes that polypeptide; (b) culturing the host cells under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing antibodies to the polypeptide.
  • a monoclonal antibody of the presently disclosed subject matter can be readily prepared through use of well-known techniques such as the hybridoma techniques exemplified in U.S. Patent No 4,196,265 and the phage-displayed techniques disclosed in U.S. Patent No. 5,260,203, the contents of which are herein incorporated by reference.
  • a typical technique involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide or polynucleotide of the presently disclosed subject matter) in a manner sufficient to provide an immune response. Rodents such as mice and rats are representative animals. Spleen cells from the immunized animal are then fused with cells of an immortal myeloma cell. Where the immunized animal is a mouse, a representative myeloma cell is a murine NS-1 myeloma cell. The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells.
  • a selected antigen e.g., a polypeptide or polynucleotide of the presently disclosed subject matter
  • Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supematants for reactivity with antigen- polypeptides. The selected clones can then be propagated indefinitely to provide the monoclonal antibody.
  • mice are injected intraperitoneally with between about 1-200 ⁇ g of an antigen comprising a polypeptide of the presently disclosed subject matter.
  • B lymphocyte cells are stimulated to grow by injecting the antigen in association with an adjuvant such as complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis).
  • an adjuvant such as complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis).
  • mice are boosted by injection with a second dose of the antigen mixed with incomplete Freund's adjuvant.
  • mice are tail bled and the sera titered by immunoprecipitation against radiolabeled antigen.
  • the process of boosting and titering can be repeated until a suitable titer is achieved.
  • the spleen of the mouse with the highest titer is removed and the spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
  • myeloma cells are obtained from laboratory animals in which such cells have been induced to grow by a variety of well-known methods. Myeloma cells lack the salvage pathway of nucleotide biosynthesis. Because myeloma cells are tumor cells, they can be propagated indefinitely in tissue culture, and are thus "immortal”. Numerous cultured cell lines of myeloma cells from mice and rats, such as murine NS-1 myeloma cells, have been established.
  • Myeloma cells are combined under conditions appropriate to foster fusion with the normal antibody-producing cells from the spleen of the mouse or rat injected with the antigen/polypeptide of the presently disclosed subject matter. Fusion conditions include, for example, the presence of polyethylene glycol.
  • the resulting fused cells are hybridoma cells.
  • hybridoma cells grow indefinitely in culture.
  • Hybridoma cells are separated from unfused myeloma cells by culturing in a selection medium such as HAT media (hypoxanthine, aminopterin, and thymidine).
  • Unfused myeloma cells lack the enzymes necessary to synthesize nucleotides from the salvage pathway because they are killed in the presence of aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not continue to grow in tissue culture. Thus, only cells that have successfully fused (hybridoma cells) can grow in the selection media.
  • Each of the surviving hybridoma cells produces a single antibody. These cells are then screened for the production of the specific antibody immunoreactive with an antigen/polypeptide of the presently disclosed subject matter.
  • Single cell hybridomas are isolated by limiting dilutions of the hybridomas. The hybridomas are serially diluted many times and, after the dilutions are allowed to grow, the supernatant is tested for the presence of the monoclonal antibody. The clones producing that antibody are then cultured in large amounts to produce an antibody of the presently disclosed subject matter in convenient quantity.
  • polypeptides and polynucleotide of the presently disclosed subject matter can be recognized as antigens, and thus identified. Once identified, those polypeptides and polynucleotide can be isolated and purified by techniques such as antibody-affinity chromatography. In antibody-affinity chromatography, a monoclonal antibody is bound to a solid substrate and exposed to a solution containing the desired antigen. The antigen is removed from the solution through an immunospecific reaction with the bound antibody. The polypeptide or polynucleotide is then easily removed from the substrate and purified.
  • the presently disclosed subject matter provides a process of detecting a polypeptide of the presently disclosed subject matter, wherein the process comprises immunoreacting the polypeptides with antibodies prepared according to the process described above to form antibody- polypeptide conjugates, and detecting the conjugates.
  • the presently disclosed subject matter provides a process of detecting messenger RNA transcripts that encode a polypeptide of the presently disclosed subject matter, wherein the process comprises hybridizing the messenger RNA transcripts with polynucleotide sequences that encode the polypeptide to form duplexes; and detecting the duplex.
  • the presently disclosed subject matter provides a process of detecting DNA molecules that encode a polypeptide of the presently disclosed subject matter, wherein the process comprises hybridizing DNA molecules with a polynucleotide that encodes that polypeptide to form duplexes; and detecting the duplexes.
  • MOR-1 K encoding polypeptides and nucleic acids can be readily used in clinical setting as a prognostic and/or diagnostic indicator for screening for levels of expression of 6TM mu-opioid receptor isoforms, or alterations in native sequences.
  • a biological sample to be screened can be a biological fluid such as extracellular or intracellular fluid, or a cell or tissue extract or homogenate.
  • a biological sample can also be an isolated cell (e.g., in culture) or a collection of cells such as in a tissue sample or histology sample.
  • a tissue sample can be suspended in a liquid medium or fixed onto a solid support such as a microscope slide.
  • a biological sample is exposed to an antibody immunoreactive with the polypeptide whose presence is being assayed.
  • exposure is accomplished by forming an admixture in a liquid medium that contains both the antibody and the candidate polypeptide.
  • Either the antibody or the sample with the polypeptide can be affixed to a solid support (e.g., a column or a microtiter plate). Additional details of methods for such assays are known in the art.
  • the presence of polypeptide in the sample is detected by evaluating the formation and presence of antibody-polypeptide conjugates. Techniques for detecting such antibody- antigen conjugates or complexes are well known in the art and include but are not limited to centrifugation, affinity chromatography and the like, and binding of a secondary antibody to the antibody-candidate receptor complex.
  • detection is accomplished by detecting an indicator affixed to the antibody.
  • exemplary and well-known indicators include radioactive labels (e.g., 32 P, 125 1, 14 C), a second antibody or an enzyme such as horseradish peroxidase. Techniques for affixing indicators to antibodies are known in the art.
  • the presently disclosed subject matter provides a process of screening a biological sample for the presence of antibodies immunoreactive with a 6TM mu-opioid receptor polypeptide.
  • a DNA or RNA molecule and particularly a DNA segment or polynucleotide can be used for hybridization to a DNA or RNA source or sample suspected of encoding a 6TM mu-opioid receptor; such molecules are referred to as "probes," and such hybridization is "probing".
  • probes can be made synthetically. The probing is usually accomplished by hybridizing the oligonucleotide to a DNA source suspected of possessing a MOR-1 K gene product.
  • the probes constitute only a single probe, and in others, the probes constitute a collection of probes based on a certain amino acid sequence or sequences of the polypeptide and account in their diversity for the redundancy inherent in the genetic code.
  • a suitable source to examine is capable of expressing a polypeptide of the presently disclosed subject matter and can be a genomic library of a cell line of interest.
  • a source of DNA or RNA can include total DNA or RNA from the cell line of interest.
  • DNA molecules can be used in a number of techniques including their use as: (1) diagnostic tools to detect sequences in DNA derived from patient's cells; (2) reagents for detecting and isolating other members of the polypeptide family and related polypeptides from a DNA library potentially containing such sequences; and (3) primers for hybridizing to related sequences for the purpose of amplifying those sequences.
  • DNA sequence information provided by the presently disclosed subject matter allows for the preparation of probes that specifically hybridize to encoding sequences of a selected MOR-1 K gene product.
  • probes of an appropriate length are prepared based on a consideration of the encoding sequence for a polypeptide of the presently disclosed subject matter. The ability of such probes to specifically hybridize to other encoding sequences lends them particular utility in a variety of embodiments.
  • the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample.
  • other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • Primers of the presently disclosed subject matter are designed to be “substantially” complementary to each strand of the genomic locus to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' sequences flanking the transition to hybridize therewith and permit amplification of the genomic locus.
  • Oligonucleotide primers of the presently disclosed subject matter are employed in the amplification method that is an enzymatic chain reaction that produces exponential quantities of polymorphic locus relative to the number of reaction steps involved.
  • one primer is complementary to the negative (-) strand of the polymorphic locus and the other is complementary to the positive (+) strand.
  • Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA polymerase I (Klenow) and nucleotides results in newly synthesized + and - strands containing the target polymorphic locus sequence.
  • the product of the chain reaction is a discreet nucleic acid duplex with termini corresponding to the ends of the specific primers employed.
  • oligonucleotide primers of the presently disclosed subject matter can be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof.
  • diethylphosphoramidites are used as starting materials and can be synthesized as described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1862.
  • One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
  • nucleic acid specimen in purified or non-purified form, can be utilized as the starting nucleic acid or acids, providing it contains, or is suspected of containing, a nucleic acid sequence containing the polymorphic locus.
  • the method can amplify, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA can be single stranded or double stranded.
  • RNA is to be used as a template, enzymes, and/or conditions optimal for reverse transcribing the template to DNA would be utilized.
  • a DNA-RNA hybrid that contains one strand of each can be utilized.
  • a mixture of nucleic acids can also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers can be so utilized.
  • the specific nucleic acid sequence to be amplified i.e., the polymorphic locus, can be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified is present initially in a pure form; it can be a minor fraction of a complex mixture, such as contained in whole human DNA.
  • DNA utilized herein can be extracted from a body sample, such as blood, tissue material (e.g., brain or kidney tissue), and the like by a variety of techniques such as that described by Maniatis et. al. (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, New York. If the extracted sample is impure, it can be treated before amplification with an amount of a reagent effective to open the cells, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily.
  • tissue material e.g., brain or kidney tissue
  • DNA utilized herein can be extracted from a body sample, such as blood, tissue material (e.g., brain or kidney tissue), and the like by a variety of techniques such as that described by Maniatis et. al. (1982) in Molecular Cloning
  • the deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90-100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization”), and the reaction is allowed to occur under conditions known in the art.
  • agent for polymerization can also be added together with the other reagents if it is heat stable.
  • This synthesis (or amplification) reaction can occur at room temperature up to a temperature above which the agent for polymerization no longer functions.
  • the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature.
  • the agent for polymerization can be any compound or system that will function to accomplish the synthesis of primer extension products, including enzymes.
  • Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase, polymerase muteins, reverse transcriptase, other enzymes, including heat-stable enzymes ⁇ i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation), such as Taq polymerase.
  • Suitable enzyme will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each polymorphic locus nucleic acid strand.
  • the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths.
  • the newly synthesized strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described herein and this hybrid is used in subsequent steps of the method.
  • the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules.
  • the steps of denaturing, annealing, and extension product synthesis can be repeated as often as needed to amplify the target polymorphic locus nucleic acid sequence to the extent necessary for detection.
  • the amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion. See McPherson et al., eds. (1991) PCR. A Practical Approach, IRL Press, Oxford University Press, New York, New York.
  • the amplification products can be detected by Southern blot analysis with or without using radioactive probes. In one such method, for example, a small sample of DNA containing a very low level of the nucleic acid sequence of the polymorphic locus is amplified, and analyzed via a Southern blotting technique or similarly, using dot blot analysis.
  • probes used to detect the amplified products can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.
  • a radioisotope for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.
  • Sequences amplified by the methods of the presently disclosed subject matter can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as dideoxy sequencing, PCR, oligomer restriction (Saiki et al. (1985) Bio/Technology 3:1008-1012), allele-specific oligonucleotide (ASO) probe analysis (Conner et ah (1983) Proc Natl Acad Sci USA 80:278), oligonucleotide ligation assays (OLAs) (Landgren et al. (1988) Science 241 :1007), and the like. Molecular techniques for DNA analysis have been reviewed (Landqren et al. (1988) Science 242:229-237).
  • the method of amplifying is by PCR, as described herein and in U.S. Patent Nos. 4,683,195; 4,683,202; and 4,965,188 each of which is hereby incorporated by reference; and as is commonly used by those of ordinary skill in the art.
  • Alternative methods of amplification have been described and can also be employed as long as a MOR-1 locus amplified by PCR using primers of the presently disclosed subject matter is similarly amplified by the alternative approach.
  • Such alternative amplification systems include but are not limited to self-sustained sequence replication, which begins with a short sequence of RNA of interest and a T7 promoter.
  • Reverse transcriptase transcribes the RNA into cDNA and degrades the RNA, followed by reverse transcriptase polymerizing a second strand of DNA.
  • Another nucleic acid amplification technique is nucleic acid sequence-based amplification (NASBATM) which uses reverse transcription and T7 RNA polymerase and incorporates two primers to target its cycling scheme.
  • NASBATM amplification can begin with either DNA or RNA and finish with either, and amplifies to about 108 copies within 60 to 90 minutes.
  • nucleic acid can be amplified by ligation-activated transcription (LAT). LAT works from a single-stranded template with a single primer that is partially single-stranded and partially double-stranded.
  • Amplification is initiated by ligating a cDNA to the promoter olignucleotide and within a few hours, amplification is about 108 to about 109 fold.
  • the QB replicase system can be utilized by attaching an RNA sequence called MDV-1 to RNA complementary to a DNA sequence of interest. Upon mixing with a sample, the hybrid RNA finds its complement among the specimen's mRNAs and binds, activating the replicase to copy the tag-along sequence of interest.
  • LCR ligase chain reaction
  • the repair chain reaction (RCR) nucleic acid amplification technique uses two complementary and target-specific oligonucleotide probe pairs, thermostable polymerase and ligase, and DNA nucleotides to geometrically amplify targeted sequences.
  • a 2-base gap separates the oligo probe pairs, and the RCR fills and joins the gap, mimicking normal DNA repair.
  • Nucleic acid amplification by strand displacement activation utilizes a short primer containing a recognition site for Hinc Il with short overhang on the 5' end which binds to target DNA.
  • a DNA polymerase fills in the part of the primer opposite the overhang with sulfur-containing adenine analogs.
  • Hinc Il is added but only cuts the unmodified DNA strand.
  • a DNA polymerase that lacks 5' exonuclease activity enters at the site of the nick and begins to polymerize, displacing the initial primer strand downstream and building a new one which serves as more primer.
  • SDA produces greater than about a 10 7 -fold amplification in 2 hours at 37°C. Unlike PCR and LCR, SDA does not require instrumented temperature cycling.
  • Another amplification system useful in the method of the presently disclosed subject matter is the QB Replicase System.
  • PCR is the preferred method of amplification if the presently disclosed subject matter, these other methods can also be used.
  • the term "amplification technique" as used herein and in the claims is meant to encompass all the foregoing methods.
  • a method for identifying a subject having a polymorphism of a MOR-1 gene comprising sequencing a target nucleic acid of a sample from a subject by dideoxy sequencing, preferably following amplification of the target nucleic acid.
  • a method for identifying a subject having a polymorphism of a MOR-1 gene comprising contacting a target nucleic acid of a sample from a subject with a reagent that detects the presence of a MOR-1 polymorphism and detecting the reagent.
  • a number of hybridization methods are well known to those skilled in the art.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those of ordinary skill in the art.
  • Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C.
  • Stringent salt conditions will ordinarily be less than 1 ,00OmM, typically less than 50OmM, and preferably less than 20OmM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wethmur & Davidson (1986) J MoI Biol 31.349-370.
  • a nucleotide sequence of the presently disclosed subject matter can be used for its ability to selectively form duplex molecules with complementary stretches of a OPRM1 gene or a MOR-1 K gene product.
  • relatively stringent conditions For applications requiring a high degree of selectivity, one typically employs relatively stringent conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by 0.02M-0.15M salt at temperatures of about 50°C to about 70°C including particularly temperatures of about 55°C, about 60 0 C and about 65°C.
  • Such conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
  • nucleic acid sequence of the presently disclosed subject matter in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator reagents include radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • enzyme tags calorimetric indicator substrates are known which can be employed to provide a reagent visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • the hybridization probes described herein are useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
  • the sample containing test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
  • the selected conditions depend inter alia on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, efc.).
  • specific hybridization is detected, or even quantified, by means of the label.
  • kits can comprise a carrier having compartments to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.
  • one of the containers can comprise an amplifying reagent for amplifying a MOR-1 or MOR-1 K DNA, such as the necessary enzyme(s) and oligonucleotide primers for amplifying target DNA from the subject.
  • the presently disclosed subject matter provides assay kits for detecting the presence of a polypeptide of the presently disclosed subject matter in biological samples, where the kits comprise a first antibody capable of immunoreacting with the polypeptide.
  • the assay kits of the presently disclosed subject matter can further comprise a second container containing a second antibody that immunoreacts with the first antibody.
  • the antibodies used in the assay kits of the presently disclosed subject matter can be monoclonal antibodies.
  • the first antibody can be affixed to a solid support.
  • the first and second antibodies can comprise an indicator, such as but not limited to a radioactive label or an enzyme.
  • the presently disclosed subject matter also provides an assay kit for screening agents.
  • a kit can contain a polypeptide of the presently disclosed subject matter.
  • the kit can additionally contain reagents for detecting an interaction between an agent and a polypeptide of the presently disclosed subject matter.
  • the presently disclosed subject matter provides assay kits for detecting the presence, in biological samples, of a polynucleotide that encodes a polypeptide of the presently disclosed subject matter, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or more contiguous nucleotide bases of, as an example, any of SEQ ID NOs: 1 or 2.
  • one or more PCR probes can be used that hybridize specifically to one or more exons present in an MOR-1 K gene product.
  • one or more probes selected from SEQ ID NOS: 5-9 can be used to detect a gene product comprising exon 13 of the human mu- opioid receptor gene.
  • a probe selected from SEQ ID NOS: 5-9 can be used in combination with a probe specific for another exon present in the MOR-1 K gene products.
  • the probe of SEQ ID NOS: 5-9 can be used in combination with a probe of SEQ ID NOS: 10-17, which are each specific for one of exons 2, 4, 5, or 0/7 of the human mu-opioid receptor gene.
  • two pairs of isolated oligonucleotide primers are provided. These sets of primers are optionally derived from a MOR-1 exon.
  • the oligonucleotide primers are useful, for example, in detecting a polymorphism.
  • the primers direct amplification of a target polynucleotide prior to sequencing.
  • isolated allele specific oligonucleotides ASO are provided.
  • the allele specific oligonucleotides are also useful in detecting a polymorphism.
  • a probe can be selected for its ability to hybridize to a 5'-reguIatory region, such as a 5' promoter region for exon 13 of MOR-1 K.
  • the probe is selective for the 5'UTR of a MOR-1 K gene product. In some embodiments, the probe is selective for an allelic variant of an internal ribosome binding site (IRES) in the 5'UTR of the MOR-1 K.
  • IRS internal ribosome binding site
  • the presently disclosed subject matter provides assay kits for detecting the presence, in a biological sample, of antibodies immunoreactive with a polypeptide of the presently disclosed subject matter, the kits comprising a MOR-1 K polypeptide that immunoreacts with the antibodies.
  • 6TM mu-opioid receptor isoform modulators can be identified by: providing a test sample comprising a 6TM mu-opioid receptor isoform polypeptide; administering a test molecule to the test sample; and determining the effect of the test molecule on the activity of the polypeptide.
  • a test molecule can be any molecule having any chemical structure.
  • a test molecule can be a polypeptide, carbohydrate, lipid, amino acid, nucleic acid, fatty acid, or steroid.
  • a test molecule can be lipophilic, hydrophilic, plasma membrane permeable, or plasma membrane impermeable.
  • the presently disclosed subject matter provides several assays that can be used to identify 6TM mu-opioid receptor modulators. Such assays involve monitoring at least one of the biological responses mediated by a 6TM mu- opioid receptor in cells expressing a polypeptide having 6TM mu-opioid receptor activity such as cells containing an exogenous nucleic acid molecule that expresses a polypeptide having 6TM mu-opioid receptor activity.
  • 6TM mu-opioid receptor-mediated responses include, without limitation, changes in intracellular calcium concentration, cAMP levels, nitric oxide release, and coupling to Ga 3 protein.
  • a 6TM mu-opioid receptor agonist can be identified using an assay that monitors intracellular calcium concentration, cAMP levels, nitric oxide release, coupling to G ⁇ s protein or an combination of these measures in cells transfected with a nucleic acid molecule that expresses a polypeptide having 6TM mu-opioid receptor activity.
  • the test sample is a non-human animal and determining the effect of the test compound is estimated by assessment of pain-related behavior in the animal.
  • Intracellular calcium concentrations can be monitored using any method.
  • intracellular calcium concentrations can be monitored using a dye that detects calcium ions.
  • cells can be loaded with a fluorescent dye (e.g., fura-2) and monitored by dual emission microfluorimetry.
  • the fura-2 loading process can involve washing the cells (e.g., one to four times) with incubation medium lacking calcium. This medium can be balanced with sucrose to maintain osmolarity. After washing, the cells can be incubated (e.g., 30 minutes) with loading solution.
  • This loading solution can contain, for example, 5 ⁇ M fura-2/AM and a non-ionic/non-denaturing detergent such as Pluronic F- 127.
  • the non-ionic/non-denaturing detergent can help disperse the acetoxymethyl (AM) esters of fura-2.
  • the cells can be washed (e.g., one to four times) with, for example, PBS without calcium or magnesium to remove extracellular dye.
  • Nitric oxide (NO) release can be monitored directly or indirectly using any method.
  • a NO-specific amperometric probe can be used to measure directly the NO released from cultured cells or tissue fragments as described elsewhere (see Stefano, et al., J. Biol. Chem., 270, 30290 (1995); and Magazine, et al., J. Immunol., 156, 4845 (1996)).
  • the concentration of NO gas in solution can be measured in real-time with, for example, a DUO 18 computer data acquisition system obtained from World Precision Instruments. Briefly, the cells or tissue fragments can be placed in a superfusion chamber containing, for example, 2 ml_ PBS.
  • a micromanipulator e.g., a micromanipulator obtained from Zeiss- Eppendorff
  • the amperometric probe Prior to obtaining measurements, the amperometric probe can be calibrated by generating a standard curve using different concentrations of a nitrosothiol donor such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) obtained from Sigma (St. Louis, Missouri, United States of America).
  • SNAP S-nitroso-N-acetyl-DL-penicillamine
  • the amperometric probe can be equilibrated in the same solution (e.g., PBS) used to incubate the cells or tissue fragments during analysis.
  • PBS e.g.
  • Ga protein can be monitored directly or indirectly using any method.
  • the cells can be lysed using radioimmunoprecipitation assay (RIPA) buffer and centrifuged at maximum RPM for 20 minutes. Supernatants should be collected and used for Co- immunoprecipitation (Co-Ip) experiments. Following overnight incubation on a rotary shaker at 4°C with anti-FLAG (MOR1 K) or anti-MYC (MOR1 ) antibodies, Ip beads (Pierce Biotechnology) are added and the samples incubated for 6 hours at 4°C.
  • RIPA radioimmunoprecipitation assay
  • the samples were then centrifuged at 14000 rpm for 10 min, the supernatants should be discarded and the beads re-suspended in 50% RIPA/PBS buffer. The procedure can be repeated 3 times.
  • the samples can then be boiled and run on 12% SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare Bio-Sciences).
  • the membranes can be probed with primary antibodies [anti- Gots (K-20): sc-823, anti-Gd (C-10): sc-262 and anti-G ⁇ (T-20): sc-378; Santa Cruz Biotechnology, 1 :1 ,000] overnight at 4°C .
  • the blots can be incubated for 4 hours at room temperature with a horseradish peroxidase-conjugated secondary antibody (1 :15,000; GE Healthcare Bio-Sciences).
  • the antibody-antigen complexes can be detected using the ECL system (Amersham) and visualized with photosensitive film (Kodak).
  • Representative modulators of six transmembrane ⁇ -opioid receptor isoform levels can comprise modulators of transcription or expression.
  • Compositions that increase or decrease the transcription or expression of six transmembrane ⁇ -opioid receptor isoform-encoding genes have clinical application for the modulation of the biological activity of six transmembrane ⁇ - opioid receptor isoforms.
  • Transcription means a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene.
  • the process includes, but is not limited to the following steps: (a) the transcription initiation, (b) transcript elongation, (c) transcript splicing, (d) transcript capping, (e) transcript termination, (f) transcript polyadenylation, (g) nuclear export of the transcript, (h) transcript editing, and (i) stabilizing the transcript.
  • “Expression” generally refers to the cellular processes by which a biologically active polypeptide is produced from RNA.
  • Transcription factor means a cytoplasmic or nuclear protein which binds to such gene, or binds to an RNA transcript of such gene, or binds to another protein which binds to such gene or such RNA transcript or another protein which in turn binds to such gene or such RNA transcript, so as to thereby modulate expression of the gene. Such modulation can additionally be achieved by other mechanisms; the essence of "transcription factor for a gene” is that the level of transcription of the gene is altered in some way.
  • Such modulation can be direct, i.e., through binding of a candidate molecule directly to the nucleotide sequence, whether DNA or RNA transcript, or such modulation can be achieved via one or more intermediaries, such as proteins other than six transmembrane ⁇ -opioid receptor isoform which are affected by the candidate compound and ultimately modulate six transmembrane ⁇ -opioid receptor isoform transcription by any mechanism, including direct binding, phosphorylation or dephosphorylation, efc.
  • This method comprises contacting a cell or nucleic acid sample with a candidate compound or molecule to be tested.
  • These samples contain nucleic acids which can contain elements that modulate transcription and/or translation of a six transmembrane ⁇ -opioid receptor isoform gene, such as a promoter or putative upstream regulatory region (representative of such as disclosed herein), and a DNA sequence encoding a polypeptide which can be detected in some way.
  • the polypeptide can be described as a "reporter” or "marker.”
  • the candidate compound directly and specifically transcriptionally modulates expression of the six transmembrane ⁇ -opioid receptor isoform- encoding gene.
  • the DNA sequence is coupled to and under the control of the promoter, under conditions such that the candidate compound or molecule, if capable of acting as a transcriptional modulator of the gene encoding six transmembrane ⁇ -opioid receptor isoform, causes the polypeptide to be expressed and so produces a detectable signal, which can be assayed quantitatively and compared to an appropriate control.
  • Candidate compounds or molecules of interest can include those which increase or decrease, i.e., modulate, transcription from the regulatory region.
  • the reporter gene can encode a reporter known in the art, such as luciferase, or it can encode six transmembrane ⁇ -opioid receptor isoform.
  • the polypeptide so produced is capable of complexing with an antibody or is capable of complexing with biotin.
  • the resulting complexes can be detected by methods known in the art.
  • the detectable signal of this assay can also be provided by messenger RNA produced by transcription of said reporter gene. Exactly how the signal is produced and detected can vary and is not the subject of the presently disclosed subject matter; rather, the presently disclosed subject matter provides the nucleotide sequences and/or putative regulatory regions of six transmembrane ⁇ -opioid receptor isoform for use in such an assay.
  • the molecule to be tested in these methods can be a purified molecule, a homogenous sample, or a mixture of molecules or compounds.
  • the DNA in the cell comprises more than one modulatable transcriptional regulatory sequence.
  • This screening method comprises separately contacting each of a plurality of substantially identical samples.
  • the plurality of samples preferably comprises more than about 10 4 samples, or more preferably comprises more than about 5 x 10 4 samples.
  • LENLEAETAPLP* is intra-cellular C-terminal domain and can be substituted with different polypeptide
  • CPPTGSPS is extra-cellular N-terminal domain and can be substituted with different polypeptide and SCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQ
  • LENLEAETAPLP* is intra-cellular C-terminal domain and can be substituted with different polypeptide

Abstract

Splice variant forms of human OPRM1 and polynucleotide sequences encoding the OPRM1 splice variants are provided, including the novel sixtrans- membrane isoforms encoded by MOR-1 K1 and MOR-1 K2. Also provided are methods of screening compounds for their ability to regulate the activity of MOR-1 splice variants and of producing antibodies to the novel isoforms. Recombinant vectors, cells, and transgenic animals comprising the novel polypeptides or polynucleotides are described. Methods and assay kits for detecting the presence of a six transmembrane μ-opioid receptor isoform polypeptide are also provided.

Description

DESCRIPTION
ALTERNATIVELY-SPLICED ISOFORM OF MU-OPIOID RECEPTOR GENE WITH CELL EXCITATORY FUNCTION
RELATED APPLICATIONS The presently disclosed subject matter is based on and claims the benefit of U.S. Provisional Application Serial No. 61/189,433, filed August 19, 2008; the disclosure of which is incorporated herein by reference in its entirety.
TECHNICAL FIELD The presently disclosed subject matter describes a class of splice variant forms of the μ-opioid receptor having cell excitatory characteristics. Also described are methods of screening drug candidates to find compounds that provide analgesia free of hyperalgesia-like effects.
BACKGROUND
The mu(μ)-type opioid receptor (O PRM 1) is a member of the G-protein- coupled receptor (GPCR) family. It has an extracellular N-terminus and intracellular C-terminus, with seven membrane-spanning domains that comprise the binding pocket for exogenous drugs. Upon activation, these seven transmembrane (7TM) domain GPCRs initiate molecular changes resulting in inhibition of nerve, immune, and glial cells that play a role in the onset and maintenance of pain. OPRM1 induces analgesia via pertussis toxin (PTX)-sensitive inhibitory G protein (Gαj/0), which inhibits cAMP formation and Ca2+ conductance and activates K+ conductance, leading to hyper-polarization of cells thereby, exerting an inhibitory effect. See Crain and Shen, Pain, 84, 121-131 (2000). Nevertheless, the opposite, stimulatory effects of opiates also have been demonstrated, depending on the experimental concentration of the drug and the duration of incubation. See Crain and Shen, Pain, 84, 121-131 (2000); and Rubovitch et al., Brain Res. MoI. Brain Res., 110, 261-266 (2003). Prolonged use of opioids leads to a number of adverse side-effects including constipation, CNS depression, dependence and potentially the exacerbation of pain via a phenomenon commonly referred to as "opioid-induced hyperalgesia" (OIH). The impact of these side effects is often exacerbated by the development of tolerance to the analgesic effects of MOR agonists. Both extremely low and extremely high doses of morphine, as well as chronic administration of opioids can elicit a hyperalgesia in animal models of pain. See Crain and Shen, Brain Res., 888, 75-82 (2001); and Li et al.. Brain Res. MoI. Brain Res., 86, 56-62 (2001). Furthermore, a dual effect of opioids on cAMP formation has been reported in cell culture. See Crain and Shen, Pain, 84, 121-131 (2000); Rubovitch et al., Brain Res. MoI. Brain Res., 110, 261-266 (2003); and Wang et al.. Neuroscience, 135, 247-261 (2005). The hyperalgesia effects of opioids correlate with cellular excitation while the analgesic effect ofopioids is associated with cellular inhibition. While the molecular mechanism mediating the excitatory effects of opiates is unclear, a switch in the G protein coupling profile of the OPRM1 from Gi to both G5 and Gq, which results in cAMP accumulation (see Crain and Shen, Pain, 84, 121- 131 (2000); Rubovitch et al.. Brain Res. MoI. Brain Res., 110, 261-266 (2003); and Wang et al., Neuroscience, 135, 247-261 (2005)); as well as adenylyl cyclase (AC) activation by Gβγ has been suggested. See Wang et al., Neuroscience, 135, 247-261 (2005). The neural mechanisms that underlie these hyperalgesic effects are poorly understood, but could be associated with concentration- and time-dependent cellular excitation (Polomano et a\.,Semin. Perioper. Nurs. 10, 3-16 (2001); Polomano et al., Semin. Perioper. Nurs., 10, 159-166 (2001); Mercadaπte et al.. J. Clin. Oncol., 19, 2898-2904 (2001)), and a biphasic effect on cAMP formation and Substance P release (Polomano etal. (2001); Mercadante et al. (2001); Chernv et al., J. CHn. Oncol., 19, 2542-2554 (2001); UhI et al., Proc. Natl Acad. Sci. USA, 96, 7752-7755 (1999); Han etal., Ann. N.Y. Acad. Sci., 1025, 370-375 (2004); Mogil, Proc. Natl Acad. Sci. USA, 96, 7744-7751 (1999)). The stimulatory effects of opiates are believed to provide a molecular mechanism underlying opioid receptor-mediated hyperanalgesia, tolerance and dependence. See Crain and Shen, Pain, 84, 121-131 (2000).
The major form of OPRM1 , also called MOR-1 , binds endogenous and exogenous opioids to produce analgesia, and to mediate basal nociception as well as agonist responses (Ready. In Miller (ed), Anesthesia, Churchill, Livingstone, pp. 2323-2350 (2000); Rowlingson and Murphy. In Miller (ed), Anesthesia, Churchill, Livingston, pp. 7752-7755 (2000); Inturrisi, Clin. J. Pain, 18, S3-S13 (2002); Goldstein, J. Am. Osteopath. Assoc, 102, S15-S21 (2002)). Opioids are the most commonly prescribed analgesics for the treatment of moderate to severe clinical pain. However, a great deal of individual variation exists in the degree of opioid analgesia and adverse side- effects, which include nausea, sedation, life-threatening respiratory depression, and paradoxical hyperalgesia. MOR-1 is coded by exons 1 , 2, 3 and 4, whereas exon 1 codes for first transmembrane domain and exon 2 and 3 code for the second through the seventh transmembrane domain. See Pasternak, Neuropharmacology, 47 Suppl 1, 312-323 (2004). There is growing evidence from rodent studies that could suggest a role of alternatively-spliced forms of OPRM1 in mediating opiate analgesia. |d- The synergistic activities of these splice variants has been proposed to explain the complex pharmacology of μ- opioids, jd- Yet, it is unclear whether the findings from the rodent studies are applicable to human opioid responses because there is a striking discrepancy between the genomic organization of mouse OPRM1 and the genomic organization of human OPRM1. In accordance with the NCBI database, the mouse OPRM1 gene includes 20 exons and codes for 41 alternative-spliced forms, while the human OPRM 1 gene includes only 9 exons and codes for only 19 alternative-spliced forms. See Unigene databases; Pasternak, Neuropharmacology, 47 Suppl 1, 312-323 (2004); Pan, DNA Cell Biol., 24, 736-750 (2005); Kvam et al., J. MoI. Med., 82, 250-255 (2004); and Doyle etal., Gene, 388, 135-147 (2007). For the majority of exons of the mouse OPRM 1 gene there are no human homologues. There are two common splicing patterns of OPRM 1 in mouse that involve the C-terminus and N-terminus. C-terminus variants contain exons 1 , 2 and 3 and code for all seven transmembrane domains, but differ structurally and functionally at the intracellular domain, a region important in transduction of the signal following receptor activation. The mouse also has a number of variants that differ in their N-terminus, some of which encode for truncated receptors. Reported mouse N-terminus variants are initiated from exon 11. Exon 11 is located approximately 10 kb upstream of exon 1 and is under the control of a different upstream promoter, suggesting alternative regulation of transcription. Three of these variants are predicted to code for truncated receptors with only six transmembrane domains (6TM), lacking TM1. The functional significance of truncated receptors is not clear, but in other receptor systems, they have been reported to modulate the activity of the full version of the receptor (see Zhu and Wess, Biochem., 37, 15773-15784 (1998); Nag et al.. Biochem. Biophys. Res. Commun. 362, 1037-1043 (2007); and Karpa et al., MoI. Pharmacol., 58, 677-683 (2000)) or change biological activity of the protein, sometime to the opposite. See Boise, et al., Ce//, 74, 597-608 (1993).
Recently, cloning of a human OPRM1 isoform, MOR-3, which is selective for opiate alkaloids and is insensitive to opioid peptides, has been reported. At the functional level, COS-1 cells transfected with the MOR-3 receptor cDNA exhibited a dose-dependent release of nitric oxide (NO) following treatment with morphine, but not with the opioid peptide DAMGO. Opioid receptor blockade with naloxone blocks the effect of morphine on COS-1 transfected cells. See Cadet et al., J. Immunol., 170, 5118-5123 (2003). This isoform lacks an amino acid sequence of approximately 90 amino acids that constitutes the extracellular N-terminal and first transmembrane TMH 1 domains and part of the first intracellular loop, but retains the ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains. Additionally, the receptor proteins are terminated by intracellular C-terminal amino acid sequences that have been hypothesized to serve as coupling or docking domains required for constitutive NO synthase (NOS) activation, which is unique among OPRM 1 isoforms. See Kream, et al., Med. Sci. Monit, 13, SC5-SC6 (2007). Northern blot and RT-PCR results showed the expression of this MOR-3 variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells. See Cadet et al., J. Immunol., 170, 5118-5123 (2003). However, uncertainty remains as to aspects of the role(s) of OPRM1 and as to the possibility of the existence of additional splice variants. Further investigation of OPRM 1 thus represents a long-felt and ongoing need in the art. SUMMARY
This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
The presently disclosed subject matter provides a class of splice variant forms of human OPRM1 and polynucleotide sequences encoding the OPRM1 splice variants. More particularly, the presently disclosed subject matter relates to exon-13 containing 6TM MOR-1 K isoforms of human OPRM1 , including alternative splice variants MOR-1 K1 and MOR-1 K2.
In some embodiments of the presently disclosed subject matter, an isolated nucleic acid sequence consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 1 , 2 or a nucleic acid sequence having at least a 98% sequence homology to SEQ ID NO: 1 or 2 over its entire length is provided.
In other embodiments, an isolated nucleic acid sequence is provided which codes for a protein consisting essentially of an amino acid sequence of
SEQ ID NO: 3 or for a protein comprising amino acids 1-300 of SEQ ID NO: 3.
Other embodiments of the presently disclosed subject matter include a recombinant vector or a recombinant host cell comprising any of the nucleic acid sequences described above.
In another embodiment, the presently disclosed subject matter provides a nucleic acid sequence that is at least 97% identical to a contiguous 902 nucleotide sequence selected from nucleotides 1309-2211 of SEQ ID NO: 1 or nucleotides 1290-2192 of SEQ ID NO: 2.
Another embodiment provides a substantially pure polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 3 or a sequence having a 97% or greater sequence homology to SEQ ID NO: 3 over its entire length; or of amino acids 1-300 of SEQ ID NO: 3 or a sequence having at least a 97% sequence homology to the sequence comprising amino acids 1-300 of SEQ ID NO: 3 over its entire length.
In another embodiment, the presently disclosed subject matter includes a recombinant cell expressing any of the polypeptides described above.
The presently disclosed subject matter further relates to methods of screening compounds for their ability to regulate the activity of MOR-1 splice variants.
In some embodiments of the presently disclosed subject matter, a method of screening candidate substances for an ability to modulate activity of a six-transmembrane μ-opioid receptor isoform is provided. The method comprises providing a test sample comprising any or the polypeptides described above, administering a test molecule to the test sample, and determining the effect of the test molecule on the activity of the polypeptide. In some embodiments, the test molecule is selected from the group consisting of a polypeptide, a nucleic acid oligonucleotide, an exogenous vector coding for a nucleic acid oligonucleotide or polypeptide, a carbohydrate, a lipid, an amino acid, a fatty acid, a steroid, and a low molecular weight organic molecule. In some embodiments, determining the effect of the test molecule on the activity of the polypeptide comprises measuring a first activity level of the polypeptide prior to administering the test molecule to the test sample, measuring a second activity level of the polypeptide after administering the test molecule to the test sample, and comparing the first and second activity levels. In further embodiments, the above method further comprises determining the effect of the test molecule on the activity of a polypeptide comprising the sequence of SEQ ID NO: 4 or amino acids 67-330 of SEQ ID NO: 4 and comparing said effect with the effect of the test molecule on the activity of any of the polypeptides of the embodiments described above. In some embodiments, the test sample is a cell. The presently disclosed subject matter includes methods wherein the polypeptide is provided to the cell from an exogenous source or wherein the cell expresses the polypeptide. In some embodiments the cell is a recombinant cell. In some embodiments, determining the effect of the test molecule comprises measuring an amount or a change in the amount of one or more of the group consisting of cAMP, calcium, nitric oxide or coupling to Gαs protein in the test sample. In some embodiments, the test sample is a non-human animal. The presently disclosed subject matter includes methods where determining the effect of the test compound is estimated by assessment of pain-related behavior in the animal. Also included are methods wherein the animal is a genetically modified animal. The genetically modified animal may possess modulated six transmembrane μ-opioid receptor isoform activity, modulated seven transmembrane μ-opioid receptor isoform activity, or a combination thereof. Also included are methods wherein the transgenic animal overexpresses any of the polypeptide embodiments described above. The genetically modified animal may be a knock-out animal that under expresses a seven transmembrane μ-opioid receptor isoform. The method includes cases where the under-expressed 7TM isoform is a polypeptide comprising the sequence of SEQ ID NO: 4 or amino acids 67-330 of SEQ ID NO: 4.
In some embodiments a transgenic non-human animal is provided, wherein the transgenic non-human animal overexpresses a six-transmembrane μ-opioid receptor isoform comprising a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology to amino acids 1-300 of SEQ ID NO: 3 over the entire length of the amino acid sequence.
In yet another embodiment, a method for identifying an antagonist or an agonist specific for a six transmembrane μ-opioid receptor isoform is provided, wherein the isoform comprises a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology thereto over the entire length of the amino acid, the method comprising contacting a cell with a test molecule and a μ- opioid receptor agonist or antagonist, wherein said cell comprises a nucleic acid molecule consisting essentially of one of SEQ ID NOs: 1 or 2 or a sequence having at least a 98% homology thereto, and wherein said cell expresses said polypeptide; and determining whether or not said test molecule reduces or prevents a non-analgesic or hyperanalgesic μ-opioid receptor- mediated response induced by said μ-opioid receptor agonist or antagonist.
In another embodiment, a method of producing an antibody immunoreactive with a six transmembrane μ-opioid receptor isoform polypeptide is provided, the method comprising steps of transfecting a recombinant host cell with a nucleic acid molecule consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 1 , 2 or a nucleic acid sequence having at least a 98% sequence homology to SEQ ID NO: 1 or 2 over its entire length or a nucleic acid sequence coding for a protein consisting essentially of an amino acid sequence of SEQ ID NO: 3 or for a protein comprising amino acids 1-300 of SEQ ID NO: 3, wherein the nucleic acid molecule encodes a six transmembrane μ-opioid receptor isoform polypeptide; culturing the host cell under conditions sufficient for expression of the polypeptide; recovering the polypeptide; and preparing an antibody to the polypeptide.
In yet another embodiment, a method of detecting a six transmembrane μ-opioid receptor polypeptide is provided, the method comprising immunoreacting the polypeptide with an antibody prepared according to the method described above to form an antibody-polypeptide conjugate; and detecting the conjugate.
The presently disclosed subject matter futher includes a method of detecting a nucleic acid molecule that encodes a six transmembrane μ-opioid receptor isoform polypeptide in a biological sample containing nucleic acid material, the method comprising the steps of hybridizing the nucleic acid molecule having a sequence complementary to one of SEQ ID NO: 1 or SEQ ID NO: 2 under stringent hybridization conditions to the nucleic acid material of the biological sample, thereby forming a hybridization duplex, and detecting the hybridization duplex.
In another embodiment an assay kit is provided for detecting the presence of a six transmembrane μ-opioid receptor isoform in a biological sample, the kit comprising a first container comprising a first antibody capable of immunoreacting with a polypeptide of any of the embodiments described above. In another embodiment, the kit further comprises a second container containing a second antibody that immunoreacts with the first antibody. In another embodiment, the first and second antibodies comprise monoclonal antibodies. In some embodiments, the first antibody is affixed to a solid support. In another embodiment, the first and second antibodies each comprise an indicator. In some embodiments, the indicator is a radioactive label or an enzyme.
In another embodiment, an assay kit is provided for detecting the presence, in a biological sample, of an antibody immunoreactive with a six transmembrane μ-opioid receptor isoform polypeptide, the kit comprising a polypeptide of any of the embodiments described above that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
In yet another embodiment, an assay kit is provided for detecting the presence, in biological samples, of a six transmembrane μ-opioid receptor isoform polypeptide, the kit comprising a first container that contains a nucleic acid molecule identical or complementary to a segment of at least ten contiguous nucleotide bases of the nucleic acid molecule consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 1 , 2 or a nucleic acid sequence having at least a 98% sequence homology to SEQ ID NO: 1 or 2 over its entire length or a nucleic acid sequence coding for a protein consisting essentially of an amino acid sequence of SEQ ID NO: 3 or for a protein comprising amino acids 1-300 of SEQ ID NO: 3.
It is an object of the presently disclosed subject matter to provide a splice variant of MOR-1 that encodes a novel 6TM receptor and methods of screening drug candidate compounds using the novel 6TM receptor.
An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying drawings as best described hereinbelow. BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 A-1 F are a series of images showing the expression pattern of exon 13 containing 0PRM1 gene splice variant in human and mouse.
Figure 1 A is an ethidium bromide gel analysis of RT-PCR performed on total RNA samples from the human brain regions known to express OPRM1 with hU2 (SEQ ID NO: 6) and hL5 (SEQ ID NO: 10) primers specific for exon
13 and exon 2. The exon 13 containing mu-opioid receptor (OPRM 1) gene splice variant MOR-1 K was detected in CNS but not in peripheral leukocytes even after a secondary PCR round with nested PCR primers. The PCR product size was 1229 nucleotides (nt), which was 3 times longer than the predicted
385 nt based on homology with the mouse genome.
Figure 1 B is an ethidium bromide gel analysis of RT-PCR analysis of mouse spinal cord with primer pairs mU2-mL3 (SEQ ID NOS: 18 and 21), and mU2-ml_1 (SEQ ID NOS: 18 and 20). A longer mouse isoform orthologous to human exon 13 was below the level of detection even by secondary PCR with the nested PCR primers mU3-ml_3 (SEQ ID NOS: 19 and 21), mU3-mL1 (SEQ ID NOS: 19 and 20) or mU3-mL2.
Figure 1 C is a schematic diagram illustrating the relative position of PCR primers designed to amplify the new alternative MOR-1 K variant in mouse and human.
Figure 1 D is a schematic diagram illustrating the exonic composition and relative position of PCR primers designed to amplify the major MOR-1 variant and the newly identified alternative MOR-1 K variant. The arrows indicate relative position of translation initiation start codons and stop codons. Figure 1 E is a schematic illustration of the predicted protein structure of
MOR-1 and MOR-1 K isoforms. Translation of the MOR-1 K variants results in a 6TM domain receptor, truncated at the N-terminus.
Figure 1 F is an ethidium bromide gel analysis of RT-PCR results demonstrating the relative expression pattern of human MOR-1 (primers hU1- L3) and MOR-1 K (primers hU5-L3; SEQ ID NOS: 5 and 11) variants. GAP3DH was used as a control for cDNA loading. All major PCR products resulting from this reaction were sequenced and aligned with human or mouse genomes. Figure 2 is a bar graph showing the expression pattern of the MOR-1 K splice variant (primers hU2-L5) relative to canonical MOR-1 isoform (primers hU1-L5, SEQ ID NOS: 5 and 11) using real time PCR approach. RT-PCR was performed on total RNA samples from the human brain regions known to express OPRM1 with primers specific for exon 1 and 2 and thus canonical MOR-1 isoform (left Y axis) and primers specific for exons 13 and exon 2 the new MOR-1 K isoform (right Y axis)( Sora et al., Proc. Natl Acad. Sci. USA, 94, 1544-1549 (1997). GAP3DH was used as a control for cDNA synthesis and PCR efficiency. Figures 3A, 3B1 and 4 are a set of graphs illustrating the cyclic adenosine monophosphate (cAMP)(Fig. 3A) and calcium (Ca2+)(FJg. 3B,4) response evoked by seven transmembrane (7TM) and six transmembrane (6TM) mu-opioid receptor (OPRM1) splice variants transiently transfected into COS1 and Be2C cells. COS1 (Fig. 3A1B) or BE2C (Fig.4) cells were transiently transfected with empty vector (Control), MOR1 (7TM) or MOR1 K (6TM) expressing constructs.
Figure 3A demonstrates that Forskoline (FSK, 10 μM) was used to increase cAMP levels prior to morphine treatment, which generally produced an inhibition of cAMP accumulation. COS1 cells transfected with MOR1 showed decreased forskolin (FSK)-induced cAMP accumulation relative to cells transfected with empty vector following morphine treatment. In contrast, cells transfected with the MOR-1 K construct showed an increase in cAMP accumulation following morphine treatment, especially prominent in non-FSK- induced condition ( last bar). Figure 3B is a bar graph showing that COS1 cells transfected with
MOR1 or empty vector did not exhibit morphine-induced changes in intracellular Ca++. In contrast, cells transfected with the MOR-1 K construct showed an increase in intracellular Ca++ release following morphine treatment.
Figure 4 is a graph showing that Be2C cells transfected with MOR1 or empty vector did not exhibit morphine-induced changes in intracellular Ca++. I n contrast, cells transfected with the MOR-1 K construct showed a dose- dependent increase in intracellular Ca++ release following morphine treatment which were antagonized in the presence of the specific MOR1 antagonist naloxone (NaI) (0.1 μM). Data are Mean + SEM, *P< 0.05.Data are reported as mean ± S.E.M from at least 6 experiments. * represent statistical significance different from the control vector condition. # represents a significant difference between the control vector condition and MOR1K transfected cells. All naloxone treatments were significantly different from the respective controls.
ANOVA was used to assess main effects followed by post hoc testing with
Tukey-Kramer method. Data are presented as mean + SEM, # and *P < 0.05.
Figures 5A-5D are graphs showing nitric oxide (NO) production evoked by 7TM and 6TM mu-opioid receptor (OPRM 1) splice variants transiently transfected into cells. BE2C cells were transiently transfected with MOR1K (6TM) or MOR1 (7TM) expressing constructs. Figure 5A shows NO production in cells transfected with MOR1K in response to increasing concentrations of morphine or the OPRM1 antagonist naloxone (NaI). The 6TM isoform (Fig. 5A), but not 7TM isoform (Fig.5B) produced robust concentration-dependent increases in the release of NO. Figures 5C-D show that the time course response associated with morphine-induced NO release was markedly different when stimulating 6TM (Fig. 5C) and 7TM (Fig. 5D) isoforms. NO release was blocked by pretreatment with naloxone (0.1 μM) and was significantly different from the respective controls (not shown). ANOVA was used to determined main effects followed by post hoc testing using Tukey-Kramer method. Data are presented as mean + SEM, # and *P < 0.05.
Figure 6 is an immunoblotting of a set of pull-down assays. The 6TM MOR-1 K Isoform couples to Gαs (top left frame). COS1 cells were transiently transfected with 7TM-MYC or 6TM-FLAG constructs. (A) lmmunoprecipitation was performed with anti-FLAG (raised in rabbit) and anti-MYC (raised in rabbit) antibodies. Immunoblotting was conducted with specific anti-Gα antibodies as described. The 6TM-FLAG construct (MOR1 K) was found to couple to Gαs, and the 7TM-MYC construct (MOR1) is coupled only to Gay0 (two center bottom frames). Consistent with Gαs binding to the 6TM isoform, morphine-mediated increases in Ca2+ levels were not prevented by pre-treatment of the cells with pertussis (PTX) in 6TM-FLAG (far right frame).
Figure 7 is a set of graphs showing that mice that develop opioid- induced hyperalgesia exhibit increased spinal MOR-1 K transcript levels. To test the hypothesis that MOR-1 K plays a role in opioid-induced hyperalgesia by being upregulated following chronic opioid administration, C57BI/6J mice were treated with morphine (10-40 mg/kg) twice daily for 4 days and MOR-1 K RNA expression was measured at the time of peak hyperalgesia. Following administration of morphine on days 1-4, mice showed analgesia, such that (A) paw withdrawal frequency to a normally noxious punctuate mechanical stimulus and (B) the number of paw flicks and jumps on a 52.5°C hotplate were significantly decreased. However, on day 5 mice showed hyperalgesia, such that responses to mechanical and thermal stimuli were significantly increased. Immediately following assessment of pain behavior on day 5, mice were sacrificed and spinal cord tissue collected for measuring transcript levels using quantitative real-time PCR. (C) Relative to vehicle-treated mice, morphine- treated mice did not exhibit changes in MOR1 RNA expression. (D) They did, however, exhibit a significant 2-fold increase in MOR-1 K RNA expression. Data are Mean + SEM, *P < 0.05.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO: 1 is a nucleotide sequence for homo sapiens μ-opioid receptor-K1 (MOR-1 K1). SEQ ID NO: 2 is a nucleotide sequence for homo sapiens μ-opioid receptor-K2 (MOR-1 K2).
SEQ ID NO: 3 is an amino acid sequence for the six transmembrane (6TM) isoform of μ-opioid receptor encoded by MOR-1 K1 and MOR-1 K2.
SEQ ID NO: 4 is an amino acid sequence for the seven transmembrane (7TM) isoform of μ-opioid receptor (GENBANK™ Accession No. P35372).
SEQ ID NO: 5 is a nucleotide sequence for hU5, an upstream PCR primer for the human μ-opioid receptor exon 13 mRNA.
SEQ ID NO: 6 is a nucleotide sequence for hU2, an upstream PCR primer for human μ-opioid receptor exon 13 mRNA. SEQ ID NO: 7 is a nucleotide sequence for hi 3U-1 , an upstream PCR primer for human μ-opioid receptor exon 13 mRNA.
SEQ ID NO: 8 is a nucleotide sequence for h13U-2, an upstream PCR primer for human μ-opioid receptor exon 13 mRNA. SEQ ID NO: 9 is a nucleotide sequence for h13U-3, an upstream PCR primer for human μ-opioid receptor exon 13 mRNA.
SEQ ID NO: 10 is a nucleotide sequence for hL5, a downstream PCR primer for human μ-opioid receptor exon 2 mRNA. SEQ ID NO: 11 is a nucleotide sequence for hL-3, a downstream PCR primer for human μ-opioid receptor exon 4 mRNA.
SEQ ID NO: 12 is a nucleotide sequence for h5L-1 , a downstream PCR primer for human μ-opioid receptor exon 5 mRNA.
SEQ ID NO: 13 is a nucleotide sequence for h5L-2, a downstream PCR primer for human μ-opioid receptor exon 5 mRNA.
SEQ ID NO: 14 is a nucleotide sequence for h5L-3, a downstream PCR primer for human μ-opioid receptor exon 5 mRNA.
SEQ ID NO: 15 is a nucleotide sequence for h5L-4, a downstream PCR primer for human μ-opioid receptor exon 5 mRNA. SEQ ID NO: 16 is a nucleotide sequence for hO/7L-1 , a downstream
PCR primer for human μ-opioid receptor exon 0/7 mRNA.
SEQ ID NO: 17 is a nucleotide sequence for hO/7L-2, a downstream PCR primer for human μ-opioid receptor exon 0/7 mRNA.
SEQ ID NO: 18 is a nucleotide sequence for mU2, an upstream PCR primer for murine μ-opioid receptor exon 13 mRNA.
SEQ ID NO: 19 is a nucleotide sequence for mU3, an upstream PCR primer for murine μ-opioid receptor exon 13 mRNA.
SEQ ID NO: 20 is a nucleotide sequence for ml_1 , a downstream PCR primer for murine μ-opioid receptor exon 2 mRNA. SEQ ID NO: 21 is a nucleotide sequence for ml_3, a downstream PCR primer for murine μ-opioid receptor exon 2 mRNA.
SEQ ID NO: 22 is a nucleotide sequence for a PCR primer for MOR1.
SEQ ID NO: 23 is a nucleotide sequence for a PCR primer for MOR1.
SEQ ID NO: 24 is a nucleotide sequence for a PCR primer for MOR1 K. SEQ ID NO: 25 is a nucleotide sequence for a PCR primer for MOR1 K.
SEQ ID NO: 26 is a nucleotide sequence for hOPRM1ex13-f, an upstream PCR primer for human OPRM1 exon 13. SEQ ID NO: 27 is a nucleotide sequence for hOPRM1ex2-r, a downstream PCR primer for human OPRM1 exon 2.
SEQ ID NO: 28 is a nucleotide sequence for hOPRM1ex1-f, an upstream PCR primer for human OPRM1 exon 1. SEQ ID NO: 29 is a nucleotide sequence for hOPRM1ex2-r(1), a downstream PCR primer for human OPRM1 exon 2.
DETAILED DESCRIPTION The presently disclosed subject matter pertains to the existence of new exons within the human MOR-1. These exons contain several unexplored polymorphisms associated with individual variability in human pain sensitivity and responses to the MOR-1 agonist morphine. Importantly, these polymorphisms are situated within a newly identified MOR-1 isoform that encodes a truncated version of the classical MOR-1 (MOR-1 K), recently cloned as described herein. The presently disclosed subject matter relates to the newly discovered exon 13 within the human OPRM1 gene locus. Exon 13- containing splice variants code for a six transmembrane (6TM) mu-opioid receptor-K (MOR-1 K) isoform that appears to have biological and clinical significance. The stimulation of MOR-1 K leads to release of excitatory neuromediators and thus an excitatory cellular response, potentially mediating opioid-induced hyperalgesia and tolerance rather than analgesic states.
MOR1 K is missing the extracellular and first transmembrane domain and, in contrast to the major MOR1 isoform, MOR1K activates a stimulatory Gas pathway and thereby elevates intracellular levels of cAMP, and Ca2+, and induces release of nitric oxide (NO). The newly identified polymorphisms are associated with pain and morphine responses, their activation leads to excitatory signaling, and their RNA levels are increased in mice with opioid hyperalgesia. Therefore, the major and the alternative MOR variants mediate opposite cellular effects and thus the balance between these isoforms can influence therapeutic outcome of opioid treatment. I. Six-Transmembrane (6TM) Human Mu-Opioid Receptor (MOR-1K)
A comprehensive study using a unique set of bioinformatics approaches for comparative analysis of mouse and human genomic DNA has indicated an extended human OPRM1 gene structure, identifying 10 new potential exons and a new putative promoter. These exons were discovered in a human genetic association study that identified several single nucleotide polymorphisms (SNPs) associated with the individual variability in pain sensitivity and responses to the MOR agonist morphine. Exons carrying these functional SNPs are spliced into a OPRM1 variant named MOR1 K. From all the isoforms, the human exon 13 containing isoform, analogous to mouse MOR-1 K isoform, appears to be of particular interest due to relative position of the functional SNP rs563649 (Figure 1C) situated within the exon 13 conservation area between mouse and human genomes (see WO 07/070252, incorporated herein by reference in its entirety). SNP rs563649, which is significantly associated with pain sensitivity and morphine analgesic efficacy, is located within a structurally conserved internal ribosomal binding site (IRES) in the 5'UTR of this novel exon 13-containing isoform and affects MOR-1 K translation efficiency. Furthermore, rs563649 exhibits very strong linkage disequilibrium throughout the entire MOR-1 gene locus, thus affecting the functional contribution of the corresponding haplotype that includes other MOR- 1 SNPs. MOR-1 K RNA is enriched in spinal cord and brain. The presently disclosed subject matter demonstrates that stimulation of MOR-1 K in vitro leads to cellular excitation characterized by increased cAMP, Ca2+, and NO production and that MOR-1 K RNA expression is increased in mice exhibiting opioid-induced hyperalgesia.
The human MOR-1 K isoform codes for a novel human truncated version of OPRM1 that includes 6, rather than 7, trans-membrane (TM) domains and lacks an extracellular domain. The extracellular N-terminus and first cytoplasmic domain are missing in this isoform. Instead, it possesses a cytoplasmic N-terminus followed by 6 transmembrane domains and C-terminus that do not differ from the 7TM isoform. This truncated 6TM receptor should retain a ligand binding pocket that is distributed across the conserved TMH2, TMH3, and TMH7 domains of MOR1 (Edwards et al., (2006) Anesthesiology, 104, 1243-1248), which means that the 6TM isoform should be capable of binding MOR agonists. MOR-1 K is an example of a functional 6TM isoform. In the genetic analysis, allelic variants coding for higher MOR1K expression was associated with greater sensitivity to noxious stimuli and poorer responses to morphine (Sora 1997), which is opposite from what one would expect in response to MOR activition. Thus it is believed that 6TM MOR receptor isoform activity contributes to hyperalgesic effects of MOR agonists throughout stimulation cellular excitatory pathways.
The cellular characterization of this novel 6TM form provides experimental evidence that this receptor variant produces excitatory rather than inhibitory cellular responses, plausibly regulating and controlling the function of OPRM1 receptors. MOR-1 K codes for a truncated 6TM OPRM1 receptor with a different intracellular domain and tissue-specific distribution as compared to a MOR-3 variant reported previously (see Cadet et al., J. Immunol., 170, 5118- 5123 (2003). In further contrast to MOR-3, MOR-1 K expresses in brain tissues and neuronal cells but not in vascular tissues or leukocytes. Cellular signaling studies suggest that the truncated MOR-1 K form contributes to hyperalgesic- like rather than analgesic states and thus represents one of the molecular mechanisms underlining excitatory effect of opioids and may contribute to tolerance, dependency and opioids-induced hyperalgesia. Accordingly, it is believed that molecular and cellular characterization of 6TM OPRM1 isoforms can provide new and important insights into the complex clinical effects of opioids.
LA1. Identification of Human MOR-IK Isoforms
RT-PCR was performed using RNA isolated from the human brain tissues known to respond to opioid treatment and that express high levels of OPRM 1 (Figure 2). The forward primers were designed to predicted exon 13 and reverse primers to predicted exon 2. See Figures 1A-1C. RT-PCR results show that the human exon 13 is approximately 0.8 kb longer than the mouse exon 13 and carries alternative acceptor sites of splicing similar to OPRM1 exons 1 , 3 and 5. See Pasternak, Neuropharmacology, 47 Suppl 1, 312-323 (2004); and Pan, DNA Cell Biol., 24, 736-750 (2005). The RT-PCR results reveal MOR-1 K expression in all examined brain tissues that are known to express MOR1 and which contribute to the pharmacological effects of MOR agonists, (Fig.2), including frontal lobe, medulla oblongata, insula, nucleus accumbens, pons, spinal cord, dorsal root ganglion (DRG). Amplification of MOR-1 K isoforms from connective tissues surrounding DRG and peripheral leukocytes was not seen. Furthermore, the human neuroblastoma cell lines Be2C and SY5Y showed the highest expression of MOR-1 K isoforms while monkey kidney COS-1 and human astrocytoma H4 cell lines did not express M0R-1K. Together, these data indicate a restricted expression of M0R-1K isoforms in neuronal cells with its highest relative expression in transformed neuronal cell lines suggesting a suppression of MOR- 1 K expression in native cellular conditions.
The sequencing results of the RT-PCR product amplified from frontal lobe, nucleus accumbens, medulla oblongata and spinal cord identified a 51UTR and a partial coding region of novel OPRM1 splicing isoforms MOR-1 K1 and MOR-1 K2, which are divergent in their 3'exon boundaries sites by 12 nucleotides (nt).
To establish the coding region and the C-terminus for the MOR-1 K isoforms, RT-PCR reactions were performed with the forward primer situated at exon 13 and reverse primers located at exons 4, 7, 8 or X. Only the form containing exons 13, 2, 3 and 4 was amplified (see Figure 1D). Translation of these new isoforms seems to be initiated at the first AUG in exon 2, as all upstream reading frames code for short peptides. Both human MOR-1 K1 and MOR-1 K2 isoforms encode a truncated version of OPRM1 that lacks an extracellular N-terminal domain and transmembrane domain I, and, thus, consists of 6 transmembrane domains (6TM) instead of the classic 7TM characteristic of major OPRM 1 isoforms. This truncated 6TM receptor is believed to retain a ligand binding pocket that is distributed across the conserved TMH2, TMH3, and TMH7domains of OPRM1 (see Fowler et al., Biochem., 43, 15796-15810 (2004)), which means that 6TM isoforms should be capable of binding OPRM1 agonists. No evidence was found for the expression of an isoform containing a short exon 13 in humans, or an isoform with a long exon 13 in mice (see Figures 1A-1C). I. B. Cellular Characterization of Six Transmembrane (6TM) Mu-
Opioid Receptor (OPRM1)
Expression vectors with cloned coding regions of 7TM (MOR-1) or 6TM (MOR-1 K) OPRM1 receptor variants were transfected into immortalized cell lines in order to characterize the cellular effects of the human 6TM isoform on cAMP and Ca2+ response. The transfected cell lines included a cell line that expresses endogenous OPRM1 (i.e., the BE2C human neuroblastoma cell line) and a cell line that does not express endogenous OPRM1 (i.e., African green monkey kidney COS1 cell line). Because MOR-coupled signaling leads to the dissociation of the heterotrimeric G-protein complex, where release of the α subunit results in the inhibition of the adenylyl cyclase/cAMP pathway and the release of the βy subunits inhibits voltage-gated Ca2+ channels (VGCC)( Polomano et a\.,Semin. Perioper. Nurs. 10, 3-16 (2001); Polomano et al., Semin. Perioper. Nurs., 10, 159-166 (2001)), the cellular characterization of cAMP accumulation and intracellular Ca2+ levels was used to assess the functional effects of MOR1 activation (Polomano et al., Semin. Perioper. Nurs., 10, 159-166 (2001); Mercadante 2001 ; Pan et al., Anesthesiology, 104, 417- 425(2006); Mogil et al., Pain, 80, 67-82 (1999); Klepstad et al.. Acta Anaesthesiol. Scand., 48, 1232-1239 (2004); Rakvag et al., Pain, 116, 73-78 (2005)). In agreement with an inhibitory function of MOR1, COS1 cells transfected with the 7TM isoform demonstrate a significant decrease in forskolin induced cAMP levels following treatment with 1 μM of morphine. In contrast, COS1 cells transfected with the 6TM isoform do not show a morphine- dependent decrease in forskolin induced cAMP levels, but instead show a very substantial increase in cAMP levels that is especially apparent in the forskolin- free condition (Fig. 3A). Similarly, COS1 cells transfected with MOR1K construct showed a substantial increase in intracellular Ca2+ release following morphine treatment (Fig. 3B), while MOR1 isoform does not show morphine- induced changes in unstimulated cells with basal Ca2+ levels. Similar results were obtained in Be2C neuroblastoma cells.
Because VGCC appears to be the primary target underlying the rapid inhibitory effects of opioids (Chou et al., Acta Anaesthesiol. Scand, 50, 787- 792 (2006); Skarke et al., CHn. Pharmacol. Ther, 73, 107-121 (2003)), and morphine stimulation of the 6TM isoform seems to increase intracellular Ca2+ levels (Fig. 3B), the dose-dependent regulation of Ca2+ levels in BE2C cells transiently transfected with 6TM (Fig. 4) was examined. BE2C cells showed a robust dose-dependent morphine-induced increase in Ca2+ levels in cells transfected with the 6TM isoform; a response that was blocked by the opioid receptor antagonism with naloxone. The increase in Ca2+ levels was significantly different from the increases observed in BE2Cs transiently transfected with either the 7TM isoform or empty vector. The increases in Ca2+ levels followed by morphine treatment of BE2C cells transfected with 7TM or empty vector are likely due to the high endogenous expression of MOR1K in this cell line (Fig.2), because these increases were not observed in COS1 cells (Fig.3B) that do not express endogenous MOR1 K (Fig.2) and were sensitive to opioid receptor blockage with naloxone (Fig.4).
In agreement with the inhibitory function of MOR-1 , COS1 cells transfected with the major 7TM form of OPRM1 showed decreased forskolin- induced (FSK; 10 μM) cAMP accumulation relative to non-transfected cells in response to treatment with morphine. In contrast, COS1 cells transfected with 6TM construct showed an increase in FSK evoked cAMP accumulation. See Figure 3A. Treatment of unstimulated COS1 cells transfected with the major 7TM form of OPRM1 or an empty vector with 1 μM morphine did not change the amount of intracellular Ca++ (not shown). In contrast, COS1 cells transfected with the 6TM form of OPRM 1 showed an increased release of intracellular Ca++ after treatment with 1 μM of morphine. See Figure 3B. Very similar results were obtained using the BE2C cell line. Morphine-dependent nitric oxide (NO) production was also characterized in neuroblastoma Be2C cells. NO production is a cellular process that is believed to contribute to morphine induced hyperalgia. See Mayer, et al., Proc, Natl., Acad, ScL U.S.A., 96, 7731-7736 (1999). Furthermore, the main cellular response mediated by another reported 6TM isoform, MOR-3, is morphine-dependent NO production, morphine-induced NO release in immortalized cell lines transfected with expression vectors for either the canonical MOR1 or the newly identified MOR-1 K receptor variant (Fig.5) was measured. 6TM transfected Be2C cells showed robust NO production in response to increasing concentrations of morphine. There were marked differences between 7TM and 6TM MORs with respect to morphine-induced NO-production in the transfected Be2C cells. Activation of 6TM transfected cells were far more sensitive to morphine resulting in a substantial dose- dependent increase in NO in 6TM transfected cells, with morphine concentrations 2-3 orders of magnitude lower than that observed with 7TM isoform stimulation (Figs.5A-5D). Pretreatment with the specific OPRM 1 antagonist naloxone attenuated the increase in production of NO (Fig. 5A), resulting in a rightward shift of the dose response curve. Be2C cells transfected with major 7TM OPRM1 form also showed a significant rightward shift of the dose response curve in comparison to cells transfected with 6TM OPRM1 form (see Figure 5B). There was also a substantial difference between 7TM and 6TM isoforms with respect in the amount of time required for NO to reach its maximum level (Figures 5C-5D). In 6TM transfected cells, maximum production (16 pM) occurred at ~25 seconds post administration and returned to a base level at ~80 sec after administration of 1 μM morphine. In contrast, NO production in MOR1 expressing cells was relatively delayed in time, reaching its maximum (11 nM) at ~55 sec after administration of a higher (1 OμM) concentration of morphine. Comparable results were obtained in COS-1 cells (data not shown).
GPCR signaling results from a dissociation of the heterotrimeric G- protein complex. Uncoupling of Gαι-Gα0 causes an inhibition of adenylate cyclase (AC) resulting in a decrease in cAMP production and the release of βy subunits which inhibits Ca2+ channels leading to an inhibition of neural activity. Conversely, uncoupling of Gas and Gαq subunits results in increase levels of cAMP followed by cellular excitation. Electrophysiological studies of the effects of opioids on nociceptive-like dorsal root ganglion (DRG) neurons in culture have provided in vivo evidence that the inhibitory effects (e.g. shortening of the Ca2+-dependent component of the action potential duration and inhibition of transmitter release) are mediated by Gαj-Gαo-dependent pathways. In contrast, the excitatory effects (e.g. prolongation of the action potential duration and stimulation of transmitter release) are mediated by Gαs -dependent pathways. In contrast, the excitatory effects (e.g. prolongation of the action potential duration and stimulation of transmitter release) are mediated by Gαs - dependent pathways (Lotsch and Geisslinger, Trends MoI. Med., 11, 82-89 (2005); Matthes et al.. Nature, 383, 819-823 (1996)).
Since activation of MOR1K results in the intracellular accumulation of cAMP and Ca++, we examined whether MOR1 K couples to Gαs, instead of Ga,. A set of co-immuno- precipitation experiments was designed to elucidate the coupling partner of MOR1 K (Fig.6). As expected, the MOR1 isoform co- immuno-precipitated with Gαiand Ga0. In contrast, we detected no coupling of MOR1K to Ga proteins other than Gαs These results were consistent with the observation that treatment with pertussis toxin (PTX) didn't block MOR1 K driven increases in intracellular Ca++ (Fig.6, far right panel). No coupling with
Gαq was observed with either MOR isoform (Fig. 6, second panel from right).
Thus, the data demonstrates that stimulation of the newly identified
MOR-1 K isoform leads to cellular excitation characterized by increased levels of cAMP and Ca2+ and increased NO production. MOR-1 K codes for a truncated 6TM OPRM 1 receptor with a different intracellular domain and tissue-specific distribution as compared to to the MOR-3 variant reported previously (see Cadet et al., J. Immunol., 170, 5118-5123 (2003)). In further contrast to MOR- 3, MOR-1 K expresses in brain tissues and neuronal cells but not in vascular tissues or leukocytes, suggesting higher relevance to morphine analgesia. Furthermore and in spite of the fact that MOR-1 K lacks the unique intracellular C-terminal amino acid sequences that are characteristic for MOR-3 and which have been hypothesized to serve as coupling or docking domains required for constitutive NO synthase (NOS) activation (see Kream etal., Med. ScL Monit, 13, SC5-SC6 (2007)), MOR-1 K stimulation leads to NO production similar to MOR-3.
The results have very broad basic cell molecular and medical implications. First, they significantly contribute to the understanding of the molecular and cellular biology of MOR receptor variants and GPCRs. The MOR1 K isoform is a MOR alternatively spliced form coding for a truncated version of MOR that lacks an extracellular N-terminal domain and transmembrane domain I resulting in a 6TM rather than the classic 7TM receptor variant. The results from the presently disclosed subject matter are the first to show that 6TM receptor activation results in increases in the intracellular production of excitatory mediators (cAMP, Ca2+ and NO), while activation of 7TM results in the inhibition of cellular activity. Furthermore, immunoprecipitation experiments revealed that the 6TM MOR isoform couples to Gαs, rather than Gαi, which couples with the canonical MOR1 isoform. These data support the view that the 6TM isoforms function as an antagonist to 7TM mediated cellular events functioning as a counterbalance to the actions mediated by the canonical 7TM isoform. Because GPCRs are major targets for therapeutic drugs commonly used in clinical practice and only a few other 6TM GPCRs isoforms have been reported for histamine H3 (Narita et al., Biochem. Biophys. Res. Commun., 311, 264-266 (2003)), prostanoid (Pasternak, Neuropharmacology, 47 (Suppl. 1), 312-323 (2004)) and adrenergic alpha 1A (Pasternak et al., J. Neurochem., 91 , 881-890 (2004)) receptors, the presently disclosed subject matter indicates functional importance of 6TM truncated GPCR isoforms in GPCR signaling and drug responses.
Second, the results suggest that the 6TM isoform mediates the molecular processes that underlie Ol H and possibly the pharmacological tolerance commonly observed with repeated dosing with opioids such as morphine. As stimulation of the 6TM isoform results in the production of excitatory mediators such as cAMP, Ca2+ and NO that have been shown to contribute to OIH, the up-regulation of this isoform would lead to changing balance between 7TM and 6M relative activities and overall analgesic outcome of the MOR agonists treatment.
MOR agonists are amongst most widely used analgesics, prescribed for both acute postoperative pain and chronic pain conditions; yet, there are substantial drug induced side effects for which there is very limited understanding. Thus, the further understanding of the molecular and cellular mechanisms that contribute to the analgesic, hyperalgesic, and analgesic tolerance effects of opioids is needed. The present results provide substantial evidence that the 6TM MOR isoform is not just another alternatively-spliced form of MOR1 , but instead contributes to the net therapeutic effects of MOR agonists by facilitating the excitatory responses to opioids and potentially represents a molecular target that mediates OIH, and analgesic tolerance. The elucidation of the biological and cellular properties engaged by 6TM and 7TM MOR receptor variants, can ultimately lead to the identification and development of a new class of opioid analgesics that show a high degree of analgesic efficacy with fewer side effects. In identifying new opioid analgesics, both the agonist and antagonist effects on 6TM and 7TM MOR isoforms can be considered. The presently claimed subject matter includes a method for identifying an antagonist or an agonist specific for a 6TM μ-opioid receptor isoform. In some embodiments, the isoform comprises a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology thereto over the entire length of the amino acid. In some embodiments, the method comprises contacting a cell with a test molecule and a μ-opioid receptor agonist or antagonist, wherein said cell comprises a nucleic acid molecule consisting essentially of one of SEQ ID NOs: 1 or 2 or a sequence having at least a 98% homology thereto, and wherein said cell expresses said polypeptide; and determining whether or not said test molecule reduces or prevents a non- analgesic or hyperanalgesic μ-opioid receptor-mediated response induced by said μ-opioid receptor agonist or antagonist.
La Materials and Methods
Cloning
MOR1 K and MOR1 expression constructs were subcloned into plRES2- EGFP (Clontech Laboratories, Inc., Mountain View, California, United States of America) expression vector using Sacl/Sacll restriction sites. MOR PCR products were generated using following primers: for MOR1 TATCGAGCTCGCCACCATG-GACAGCAGCGCTGCCCCCACGAAC (SEQ ID NO: 22) and ATCCCCGCGGTT-AGGGCAACGGAGCAGTTTCTGCTTC (SEQ ID NO: 23); for MOR1K
ATGCTCTAGATTAGGGCAACGGAGCAGTTTCTGCTTC (SEQ ID NO: 24) and AAGCTTGCCACCATGAAGACTGCCACCAACATCTACATTTTC (SEQ ID NO: 25)). All constructs were sequence verified. Cell culture
BE2C and COS1 cells were obtained from ATCC. The cells we grown to 90% confluence and transfected with MOR1K, MOR1 or GFP expression constructs with Lipofectamin 2000 reagent using manufacturer guidelines.
RT-PCR analysis
The RNA was extracted using Quiagen RNAeasy kit. The isolated RNA was treated with RNase free-DNase I (Promega, Madison, Wisconsin, United States of America) and reverse transcribed byThermo-X reverse transcriptase (Invitrogen, Carlsbad, California, United States of America). The cDNA was amplified with DyNAmo-SYBRGreen qPCR kit (MJ Research, Waltham, Massachusetts, United States of America) using forward and reverse PCR primers
Primer Tm Sequence SEQ ID NO hOPRMlexl3-f 58 AGTGGTTCCCAGAGTGAAACTGA 26 hOPRMlex2-r 55 GCCAGAGCAAGGTTGAAAATG 27 hOPRMlexl-f 58 CTTCCTGGTCATGTATGTGATTGTC 28 hOPRMlex2-r(l) 58 GCCAGAGCAAGGTTGAAAATG 29
using 5OC for 2 min and 95C for 10 min followed by 40 cycles of 95C for 15 sec and 6OC for 1 min. Opticon-2 Real Time Fluorescence Detection System (MJ Research) was used for measuring fluorescence.
Calcium measurement
BE2C human neuroblastoma cells (American Type Culture Collection - ATCC) were grown to near confluence in black 96-well poly-D-lysine coated plates. The cell cultures were grown in DMEM/F12 media. The indicator Fluo-4 NW dye (Invitrogen) was prepared as outlined in manufacturer instructions. 10OμL of Fluo-4 NW dye was added to each well. The plate was then incubated with the lid on at 37°C for 30 minutes, then at room temperature for an additional 30 minutes. The fluorescence was measured using Victror-3 (Perkin Elmer, Waltham, Massachusetts, United States of America) microplate reader with settings for emission at 515 nm and excitation at 500 nm.
Nitric oxide (NO) measurement Cells were grown in DMEM/F12 supplemented with 10% FBS at 37°C.
NO release from the transfected and untransfected cell lines is directly measured using an NO-specific amperometric probe. The amperometric probe was allowed to equilibrate for at least 10 min before being transferred to the well containing the cells. Morphine-stimulated NO release was evaluated in response to increasing morphine concentrations, such as in the range of 10"5— 10"9 M. Each experiment is repeated four times along with a control (cells transfected with vector alone). CyAMP assay
For the cAMP accumulation assay, MOR or MOR1 K expressants were plated on 12 well plates and grown to 90% confluency. On the day of sample preparation, cells were washed with DMEM to remove serum and incubated with serum-free DMEM containing the phosphodiesterase inhibitor 100 μM of IBMX (Sigma-Aldrich, St. Louis, Missouri, United States of America) for 30 min, morphine was then added and cells incubated for a further 15 min. Following this, forskoline FSK (50 μM) was added to the wells and the cells were incubated for 15 min to stimulate cAMP production. DMSO alone was used as a vehicle control. After incubation, reactions were terminated by aspiration of the medium and addition of 0.1 M HCI followed by 20 min incubation at room temperature. After centrifugation of the cell samples at 10,000 g for 10 min, protein content of the supernatant was assessed and the samples were diluted to protein concentrations of 20 μg/ml the levels of cAMP were determined using enzyme immunoassay (EIA) cAMP EIA kit (Discoverex, Fremont, California, United States of America) according to the manufacturer specifications.
Co-lmmuno precipitation
The cells were lysed using RIPA buffer and centrifuged at maximum RPM for 20 minutes. Supernatants were collected and used for Co-Ip experiments. Following the overnight incubation on the rotary shaker at 4°C overnight with anti-FLAG (MOR1 K) or anti-MYC (MOR1 ) antibodies the beads (Pierce Biotechnology, Rockford, Illinois, United States of America) were added and the samples were incubated for 6 hours at 4°C. The samples were then centrifuged at 14000 rpm for 10 min, the supernatants were discarded and the beads were re-suspended in 50% RIPA/PBS buffer. The procedure was repeated 3 times. The samples were then boiled and run onto 12% SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare Bio-Sciences, Piscataway, New Jersey, United States of America). After blocking (overnight at 4°C) with 5% non-fat dried milk in Tris- buffered saline with Tween 20 (blocking buffer, TBS-T, 150 mM NaCI, 20 mM Tris-HCI, pH 7.5, 0.1% Tween 20), the membranes were probed with primary antibodies [anti-G0,s (K-20): sc-823, anti-Gd (C-10): sc-262 and anti-Gβ (T-20): sc-378; Santa Cruz Biotechnology, Santa Cruz, California, United States of America, 1 : 1 ,000] for overnight at 4°C . After washing in TBS-T (three times, 5 min each), the blots were incubated for 4 hours at room temperature with a horseradish peroxidase-conjugated secondary antibody (1 :15,000; GE Healthcare Bio-Sciences). All antibodies were diluted in blocking buffer. The antibody-antigen complexes were detected using the ECL system (Amersham, Pittsburgh, Pennsylvania, United States of America) and visualized with photosensitive film (Kodak, Rochester, New York, United States of America).
LD. Behavioral data from C57BI/6J mice
Behavioral studies demonstrated that the C57BI/6J mice up regulate the 6TM variant (MOR1K) following chronic exposure to morphine when opioid induced hyperalgesia (OIH) is present. Specifically, Figure 7 exhibits representative data showing that in C57BI/6J mice morphine suppresses both noxious mechanical and thermal pain perception and that repeated daily administration of escalading doses of morphine over a 4 day period results in marked signs of mechanical (Panel A) and thermal (Panel B) hyperalgesia. The presence of OIH was associated with increased spinal cord expression of the MOR-1 K (Panel D), but not MOR1 (Panel C). These data provide the evidence that up-regulation of 6TM isoforms contributes to morphine- dependent hyperalgesic states. Subjects
Sixteen male and sixteen female adult C57B/6J, CXBK, and 129 SV/EV mice (20-3Og; Jackson Labs, Bar Harbor, Maine, United States of America, and Taconic, Germantown, New York, United States of America) were used in these experiments. All procedures were approved by the University of North Carolina Animal Care and Use Committee and adhered to the guidelines of the Committee for Research and Ethical Issues of the IASP (Zimmermann, 1993).
Drugs and Chemicals Morphine sulfate was dissolved in 0.9% saline with volumes depending on the desired ending doses of 10 mg/kg, 20 mg/kg, or 40 mg/kg. The injection volumes were 20-30 uL depending on the weight of the animal. The morphine sulfate was obtained from the National Institute for Drug Abuse.
General experimental methods
Mice were handled and habituated to the testing environments for 4 days prior to establishing baseline responsiveness to all tests. On testing days, mice were placed in the open field infrared tracking system and their movements were tracked for a 15 minute period. The number of feces pellets produced by each mouse was noted at the conclusion of the 15 minute period.
The mice were then placed in Plexiglas™ cages positioned over an elevated perforated stainless steel platform and habituated to the environment for 10 minutes priorto the mechanical testing. Afterthe conclusion of the mechanical testing, mice were individually placed onto a hot plate and their reactions were videotaped for a period of one minute. Mice were then returned to their home cages.
In this study, the mice were baselined for one day. The mice then received either morphine or vehicle subcutaneous injections twice daily for 4 days. On day 1 , the morphine dosage was 10 mg/kg. Days 2 and 3 had a morphine dosage of 20 mg/kg and the final day had a morphine dosage of 40 mg/kg. During the baseline day and days 5-7, the behavior testing regimen was conducted once per group in the morning. During days the mice received injections, mice were tested both before and following the injection in the morning. The mice were also tested in the open field infrared tracking device prior to and following the night injection.
Assessment of motor activity and defecation rate Motor activity was evaluated using an infrared tracking system with Acti-
Track software (Panlab, Barcelona, Spain). Mice where placed into the Plexiglas™ container where they had free access to move around. Their movements were tracked for a 15 minute period and then analyzed for total distance traveled. After the mice were removed from the container, the number of feces pellets was recorded in order to determine defecation rates.
Assessment of paw withdrawal threshold, allodynic, and hyperalgesic response to mechanical stimuli
Paw withdrawal threshold was assessed using the up-down method (Chaplan et al., J Neurosci Methods 53:55-63 (1994)) to determine the threshold for punctate mechanical stimulation. A series of eight calibrated filaments (with bending forces of 0.07, 0.17, 0.41 , 0.70, 1.19, 1.50, 2.05, 3.63g; (Stoelting Co., Wood Dale, Illinois, United States of America)) were presented to the hind paw in successive order, whether ascending or descending. Filaments were positioned in contact with the hind paw for a duration of 3 s or until a withdrawal response occurred. Testing was initiated with the middle hair of the series (0.70 g). In the absence of a paw withdrawal response, an incrementally stronger filament was presented and in the event of a paw withdrawal, an incrementally weaker filament was presented. After the initial response threshold was crossed, this procedure was repeated in order to obtain a total of six responses in the immediate vicinity of the threshold. The pattern of withdrawals (X) and absence of withdrawals (O) were noted together with the terminal filament used in the series of six responses. The 50% g threshold = (10tx f +/cδ])/10,000, where Xf = value (in log units) of the final von Frey hair used; k - tabular value of pattern of positive (X) and negative (O) responses, and δ = mean difference (in log units) between stimuli.
Immediately following determination of the response threshold, paw withdrawal frequency (%) to punctate mechanical stimulation was assessed. A von Frey monofilament with a calibrated bending force of 0.40 g was presented to the hind paw ten times for a duration of 1 s with an interstimulus interval of approximately 1 s. Mechanical allodynia was defined as an increase in the percentage frequency ([No. of paw withdrawals/10] 100) of paw withdrawal evoked by stimulation with von Frey monofilaments. After the all of the animals were tested with the 0.40 g monofilament, the process was repeated with a 1.50 g force monofilament to determine mechanical hyperalgesia.
Assessment of thermal hyperalgesia Thermal hyperalgesia was evaluated using the hot plate method. Mice were placed in a hot plate behavior testing apparatus (Columbus Instruments, Columbus, Ohio, United States of America) with a plate temperature of 51.5°C for a period of one minute. Mice were videotaped for this duration and then later analyzed for behavioral actions. The number of jumps and paw flicks (any paw) were recorded for each mouse during the testing period. The number of these actions where combined to create a "total reactions" score for each animal.
\l General Considerations 11.A. Nucleic Acids
The terms "nucleic acid" and "nucleic acid sequence" as used herein encompass both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
An isolated nucleic acid of amino acid sequence is one that is substantially free of the materials with which it is associated in its native environment. In some embodiments, it is meant at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% free of such materials. In some embodiments, the term "isolated" as used herein with reference to nucleic acid sequences refers to a naturally-occurring nucleic acid sequence that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 31 end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally- occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.
The term "isolated" as used herein with reference to nucleic acids also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non- naturally-occurring nucleic acids such as an engineered nucleic acid can be considered to be isolated nucleic acid. Engineered nucleic acids can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acids can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.
The term "exogenous" as used herein with reference to nucleic acids ( or with reference to amino acid sequences or peptides) and a particular cell refers to any nucleic acid that does not originate from that particular cell as found in nature. Thus, all non-naturally-occurring nucleic acids are considered to be exogenous to a cell once introduced into the cell. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid sequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a cell once introduced into the cell, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non- naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acids since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally- occurring nucleic acid.
A nucleic acid that is naturally-occurring can be exogenous to a particular cell. For example, an entire chromosome isolated from a cell of animal X is an exogenous nucleic acid with respect to a cell of animal Y once that chromosome is introduced into Y's cell.
The presently disclosed subject matter provides for isolated nucleic acid sequences consisting essentially of a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Thus, the presently disclosed nucleic acid sequences relate to exon 13-containing splice variants of the human OPRM1 gene. In some embodiments, the nucleic acid sequences comprise exons 13, 2, 3, and 4. By "consisting essentially of is meant a sequence that is at least 95%, 96%, 97%, 98%, or 99% homologous to a given sequence, such as SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the sequence will be at least 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 1 or SEQ ID NO: 2 over the entire length of the sequence. In some embodiments, the sequence that consists essentially of SEQ ID NO: 1 or SEQ ID NO: 2 includes one nucleotide addition, deletion or substitution. In some embodiments, the nucleic acid consists of SEQ ID NO: 1 or SEQ ID NO: 2.
In some embodiments, the presently disclosed subject matter provides an isolated nucleic acid sequence that codes for a protein consisting essentially of SEQ ID NO: 3. In some embodiments, the sequence encodes for a protein comprising amino acids 1-300 of SEQ ID NO: 3 or that consists essentially of amino acids 1-300 over the entire length of the amino acid sequence.
In some embodiments, the isolated nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a contiguous sequence of the coding region of SEQ ID NO: 1 or SEQ ID NO: 2. Thus, in some embodiments, the isolated nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a contiguous 902 nucleotide sequence selected from nucleotides
1309-2211 of SEQ ID NO: 1 and nucleotides 1290-2192 of SEQ ID NO: 2.
Sequence identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group. The GAP program utilizes the alignment method of Needleman et al. (1970) J MoI Biol 48:443, as revised by Smith et al. (1981) Adv Appl Math 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. Representative parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See Schwartz et al. (1979) Nuc Acids Res 6(2):745-755; Gribskov et al. (1986) Nuc Acids Res 14(1):327-334. MOR-1 K, MOR-1 K1 , or MOR-1 K2 gene products which have functionally equivalent codons are covered by the presently disclosed subject matter. The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the ACG and AGU codons for serine. Thus, when referring to the sequence examples presented in SEQ ID NOs: 1 and 2 applicants provide substitution of functionally equivalent codons of Table 1 into the sequence examples of SEQ ID NOs: 1 and 2. Thus, applicants are in possession of amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience. TABLE 1 - Functionally Equivalent Codons
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic Acid Asp D GAC GAU
Glumatic acid GIu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine GIy G GGA GGC GGG GGU
Histidine His H CAC CAU lsoleucine lie I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine GIn Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S ACG AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine VaI V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
In some embodiments, the presently disclosed subject matter provides a nucleic acid that is complementary to the MOR-1 K splice variant (e.g., MOR- 1K1 or MOR-1 K2). Nucleic acid sequences which are "complementary" are those, which are base-paired according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein. A particular example of a provided complementary nucleic acid segment is an antisense oligonucleotide.
One technique in the art for assessing complementary sequences and/or isolating complementary nucleotide sequences is hybridization. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of about 300C, typically in excess of about 37°C, and optionally in excess of about 45°C. Stringent salt conditions will ordinarily be less than about 1 ,000 mM, typically less than about 500 mM, and optionally less than about 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See e.g. , Wethmur & Davidson (1968) J MoI Biol 31 :349-370. Determining appropriate hybridization conditions to identify and/or isolate sequences containing high levels of homology is well known in the art. See e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. For the purposes of specifying conditions of high stringency, representative conditions are salt concentration of about 200 mM and temperature of about 45°C. One example of such stringent conditions is hybridization at4XSSC, at 65°C, followed by a washing in 0.1XSSC at65°C for one hour. Another exemplary stringent hybridization scheme uses 50% formamide, 4XSSC at 42°C. As used herein, "stringent conditions" means conditions of high stringency, for example 6XSSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 μg/ml salmon sperm DNA and 15% formamide at 68°C. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M NaCI/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC. Sequence identity can be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM NaCI/0.9 mM sodium citrate).
II.B. Vectors The presently disclosed subject matter further encompasses MOR-1 K splice variant polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element if necessary.
A "vector" refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo. The heterologous polynucleotide can comprise a sequence of interest for purposes of therapy or biomedical or genetic research, and can optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors for translation of a polynucleotide encoding sequence. Also included are viral vectors.
The term "recombinant" means a polynucleotide of genomic cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.
"Heterologous" means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide, can be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter. The polynucleotides of the presently disclosed subject matter can comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct desirable to provide embodiments of the presently disclosed subject matter. A "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector.
When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof.
Polynucleotides comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Polynucleotides can be introduced into host cells by any means known in the art. The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including direct uptake, endocytosis, transfection, f-mating, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is infectious, for instance, a retroviral vector). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.
Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. Amplified DNA can be isolated from the host cell by standard methods. See, e.g., Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, incorporated herein in its entirety. RNA can also be obtained from transformed host cell, or it can be obtained directly from the DNA by using a DNA-dependent RNA polymerase. The preparation of recombinant vectors is well known to those of skill in the art and described in many references, such as, for example, Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, incorporated herein in its entirety. It is understood that the DNA coding sequences to be expressed, in this case those encoding the MOR-1 K gene products, are positioned in a vector adjacent to and under the control of a promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one generally positions the 5' end of the transcription initiation site of the transcriptional reading frame of the gene product to be expressed between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter.
One can also desire to incorporate into the transcriptional unit of the vector an appropriate polyadenylation site (e.g., 5'-AATAAA-3'), if one was not contained within the original inserted DNA. Typically, these poly-A addition sites are placed about 30 to 2000 nucleotides "downstream" of the coding sequence at a position prior to transcription termination.
While use of the control sequences of the specific gene will be preferred, other control sequences can be employed, so long as they are compatible with the genotype of the cell being treated. Thus, one can mention other useful promoters by way of example, including, e.g., an SV40 early promoter, a long terminal repeat promoter from retrovirus, an actin promoter, a heat shock promoter, a metallothionein promoter, and the like.
As is known in the art, a promoter is a region of a DNA molecule typically within about 100 nucleotide pairs upstream of (i.e., 5' to) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes.
Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer imposes specificity of time, location and expression level on a particular coding region or gene. A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. An enhancer can function when located at variable distances from transcription start sites so long as a promoter is present.
As used herein, the phrase "enhancer-promoter" means a composite unit that contains both enhancer and promoter elements. An enhancer- promoter is operatively linked to a coding sequence that encodes at least one gene product. As used herein, the phrase "operatively linked" means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Approaches for operatively linking an enhancer-promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the enhancer-promoter.
An enhancer-promoter used in a vector construct of the presently disclosed subject matter can be any enhancer-promoter that drives expression in a cell to be transfected. By employing an enhancer-promoter with well- known properties, the level and pattern of gene product expression can be optimized.
For introduction of a vector construct that will deliver the gene to the affected cells is desired. Viral vectors can be used. These vectors can be an adenoviral, a retroviral, a vaccinia viral vector, adeno-associated virus or Lentivirus; these vectors have been successfully used to deliver desired sequences to cells and tend to have a high infection efficiency. Suitable vector- target gene constructs are adapted for administration as pharmaceutical compositions, as described herein below. Viral promoters can also be of use in vectors of the presently disclosed subject matter, and are known in the art.
Commonly used viral promoters for expression vectors are derived from polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment that also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided there is included the approximately 250 base pair sequence extending from the Hind III site toward the BgI I site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
The origin of replication can be provided either by construction of the vector to include an exogenous origin, such as can be derived from SV40 or other viral source, or can be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
In an alternative embodiment, the presently disclosed subject matter provides an expression vector comprising a polynucleotide that encodes a biologically active mu-opioid receptor isoform polypeptide in accordance with the presently disclosed subject matter. Optionally, an expression vector of the presently disclosed subject matter comprises a polynucleotide that encodes a polypeptide consisting essentially of SEQ ID NO: 3 or of amino acids 1-300 of SEQ ID NO: 3. Optionally, an expression vector of the presently disclosed subject matter comprises a polynucleotide comprising the nucleotide sequence consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 2. Optionally, an expression vector of the presently disclosed subject matter comprises a polynucleotide operatively linked to an enhancer-promoter. Optionally, an expression vector of the presently disclosed subject matter comprises a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, an expression vector of the presently disclosed subject matter comprises a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter and the expression vector further comprises a polyadenylation signal that is positioned 3' of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide.
In yet another embodiment, the presently disclosed subject matter provides a recombinant host cell transfected with a polynucleotide that encodes a biologically active mu-opioid receptor isoform polypeptide in accordance with the presently disclosed subject matter. Optionally, a recombinant host cell of the presently disclosed subject matter is transfected with the polynucleotide that encodes human MOR-IK polypeptide. Optionally, a recombinant host cell of the presently disclosed subject matter is transfected with the polynucleotide sequence encoding or set forth in any of SEQ ID NOs: 1-2. Optionally, a recombinant host cell is a mammalian cell. In another aspect, a recombinant host cell of the presently disclosed subject matter is a prokaryotic host cell, including parasitic and bacterial cells. Optionally, a recombinant host cell of the presently disclosed subject matter is a bacterial cell, such as but not limited to a strain of Escherichia coli. By way of example, a recombinant host cell can comprise a polynucleotide under the transcriptional control of regulatory signals functional in the recombinant host cell, wherein the regulatory signals appropriately control expression of the mu- opioid receptor polypeptide in a manner to enable all necessary transcriptional and post-transcriptional modification. II.C. Polypeptides
The presently disclosed subject matter provides substantially pure polypeptides. The term "substantially pure" as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
Any suitable method can be used to obtain a substantially pure polypeptide. For example, common polypeptide purification techniques such as affinity chromotography and HPLC as well as polypeptide synthesis techniques can be used. In addition, any material can be used as a source to obtain a substantially pure polypeptide. For example, tissue from wild-type or transgenic animals can be used as a source material. In addition, tissue culture cells engineered to over-express a particular polypeptide of interest can be used to obtain substantially pure polypeptide. Further, a polypeptide within the scope of the presently disclosed subject matter can be engineered to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FLAG™ tag (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini. Other fusions that could be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.
The presently disclosed subject matter provides polypeptides consisting essentially of SEQ ID NO: 3. By "consisting essentially of refers to an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence homology to a give sequence, such as SEQ ID NO: 3. In some embodiments, the amino acid comprises a sequence consisting of amino acids 1-300 of SEQ ID NO: 3. In some embodiments, the amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% sequence homology to amino acids 1- 300 of SEQ ID NO: 3 over the entire length of the sequence. In some embodiments, the polypeptide consists of SEQ ID NO: 3. In some embodiments, the terms "6TM mu-opioid receptor" and "6TM mu-opioid receptor isoform" refer to a polypeptide consisting essentially of SEQ ID NO: 3 or encoded by the nucleic acid sequence consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, "7TM mu-opioid receptor" or "7TM mu-opioid receptor isoform" refer to a polypeptide consisting essentially of SEQ ID NO: 4 or comprising the sequence consisting essentially of amino acids 67- 330 of SEQ ID NO: 4.
II. D. Biologically Functional Equivalents
As mentioned above, modifications and changes can be made in the structure of the mu-opioid receptor isoform proteins and peptides described herein and still constitute a molecule having like or otherwise desirable characteristics. For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive capacity with, for example, structures in the nucleus of a cell. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or the nucleic acid sequence encoding it) to obtain a protein with the same, enhanced, or antagonistic properties. Such properties can be achieved by interaction with the normal targets of the native protein, but this need not be the case, and the biological activity is not limited to a particular mechanism of action. It is thus provided in accordance with the presently disclosed subject matter that various changes can be made in the sequence of the mu-opioid receptor isoform proteins and peptides or underlying nucleic acid sequence without appreciable loss of their biological utility or activity.
Biologically functional equivalent peptides, as used herein, are peptides in which certain, but not most or all, of the amino acids can be substituted. Thus, when referring to the nucleic acid sequence examples presented in SEQ ID NOs: 1 and 2 applicants provide substitution of codons that encode biologically equivalent amino acids as described herein into the sequence examples of SEQ ID NO: 3. Thus, applicants are in possession of amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience.
Alternatively, functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man can be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine a desired activity at the molecular level. Amino acid substitutions, such as those which might be employed in modifying the mu-opioid receptor isoform proteins and peptides described herein, are generally based on the relative similarity of the amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents. Other biologically functionally equivalent changes will be appreciated by those of skill in the art.
In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (- 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte etal. (1982) J MoI Biol 157:105, herein incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ± 2 of the original value is preferred, those, which are within ± 1 of the original value, are particularly preferred, and those within ± 0.5 of the original value are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101 , incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein.
As detailed in U.S. Patent No. 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartate (+ 3.0 ± 1); glutamate (+ 3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-
1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ± 2 of the original value is preferred, those, which are within ± 1 of the original value, are particularly preferred, and those within ± 0.5 of the original value are even more particularly preferred.
While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes can be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons can code for the same amino acid.
Thus, it will also be understood that the presently disclosed subject matter is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NOs: 1-3. Recombinant vectors and isolated DNA segments can therefore variously include the mu-opioid receptor polypeptide-encoding region itself, include coding regions bearing selected alterations or modifications in the basic coding region, or include larger polypeptides which nevertheless comprise mu-opioid receptor polypeptide-encoding regions or can encode biologically functional equivalent proteins or peptides which have variant amino acid sequences.
The presently disclosed subject matter further encompasses fusion proteins and peptides wherein the mu-opioid receptor isoform coding region is aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes.
Further, in addition to the peptidyl compounds described herein, it is also provided that other sterically similar compounds can be formulated to mimic the key portions of the peptide structure. Such compounds can be used in the same manner as the peptides of the presently disclosed subject matter and hence are also functional equivalents. The generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the presently disclosed subject matter.
II. E Transgenic Animals
It is also provided within the scope of the presently disclosed subject matter to prepare transgenic non-human animals that express a human mu- opioid receptor splice variant (e.g., MOR-1K1 or MOR-1K2) or that have modified OPRM1 expression. A representative transgenic animal is a mouse.
Techniques for the preparation of transgenic animals are known in the art. Exemplary techniques are described in U.S. Patent No. 5,489,742
(transgenic rats); U.S. Patent Nos.4,736,866, 5,550,316, 5,614,396, 5,625,125 and 5,648,061 (transgenic mice); U.S. Patent No. 5,573,933 (transgenic pigs); 5,162,215 (transgenic avian species) and U.S. Patent No. 5,741,957 (transgenic bovine species), the entire contents of each of which are herein incorporated by reference. With respect to a representative method for the preparation of a transgenic mouse, cloned recombinant or synthetic DNA sequences or DNA segments encoding a MOR-1 K gene product are injected into fertilized mouse eggs. The injected eggs are implanted in pseudo pregnant females and are grown to term to provide transgenic animals whose cells overexpress a MOR- 1 K gene product.
The presently disclosed subject matter further relates to transgenic animals with a specific "knock-out" modification. For example, the transgenic animal can be provided that under expresses a seven-transmembrane (7TM) mu-opioid receptor isoform.
In a knockout, it can be desirable for the target gene expression to be undetectable or insignificant. For example, a knockout of a target gene means that function of the gene has been substantially decreased so that expression is not detectable or only present at insignificant levels. This can be achieved by a variety of mechanisms, including introduction of a disruption of the coding sequence, e.g. insertion of one or more stop codons, insertion of a DNA fragment, etc., deletion of coding sequence, substitution of stop codons for coding sequence, etc.
Different approaches can also be used to achieve the "knockout". A chromosomal deletion of all or part of the native gene can be induced, including deletions of the non-coding regions, particularly the promoter region, 31 regulatory sequences, enhancers, or deletions of gene that activate expression of target genes. Afunctional knock-out can also be achieved by the introduction of an anti-sense construct that blocks expression of the native genes (for example, see Li and Cohen (1996) Ce// 85:319-329). "Knockouts" also include conditional knock-outs, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally. II.F. Generation of Antibodies
In still another embodiment, the presently disclosed subject matter provides an antibody immunoreactive with a polypeptide of the presently disclosed subject matter. Optionally, an antibody of the presently disclosed subject matter is a monoclonal antibody. Techniques for preparing and characterizing antibodies are well known in the art (See e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide or polynucleotide of the presently disclosed subject matter, and collecting antisera from that immunized animal.
A wide range of animal species can be used for the production of antisera.
Because of the relatively large blood volume of rabbits, a rabbit is a representative choice for production of polyclonal antibodies. As is well known in the art, a given polypeptide or polynucleotide can vary in its immunogenicity. It is often necessary therefore to couple the immunogen (e.g., a polypeptide or polynucleotide) of the presently disclosed subject matter) with a carrier. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
Approaches for conjugating a polypeptide or a polynucleotide to a carrier protein are well known in the art and include glutaraldehyde, NCmaleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis- biazotized benzidine.
As is also well known in the art, immunogenicity to a particular immunogen can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Exemplary adjuvants include complete
Freund's adjuvant, incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen used of the production of polyclonal antibodies varies, inter alia, upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen, e.g., subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal. The production of polyclonal antibodies is monitored by sampling blood of the immunized animal at various points following immunization. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored.
In another aspect, the presently disclosed subject matter provides a process of producing an antibody immunoreactive with a six transmembrane mu-opioid receptor isoform polypeptide, the process comprising the steps of (a) transfecting recombinant host cells with a polynucleotide that encodes that polypeptide; (b) culturing the host cells under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing antibodies to the polypeptide.
A monoclonal antibody of the presently disclosed subject matter can be readily prepared through use of well-known techniques such as the hybridoma techniques exemplified in U.S. Patent No 4,196,265 and the phage-displayed techniques disclosed in U.S. Patent No. 5,260,203, the contents of which are herein incorporated by reference.
A typical technique involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide or polynucleotide of the presently disclosed subject matter) in a manner sufficient to provide an immune response. Rodents such as mice and rats are representative animals. Spleen cells from the immunized animal are then fused with cells of an immortal myeloma cell. Where the immunized animal is a mouse, a representative myeloma cell is a murine NS-1 myeloma cell. The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells. Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media. This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supematants for reactivity with antigen- polypeptides. The selected clones can then be propagated indefinitely to provide the monoclonal antibody.
By way of specific example, to produce an antibody of the presently disclosed subject matter, mice are injected intraperitoneally with between about 1-200 μg of an antigen comprising a polypeptide of the presently disclosed subject matter. B lymphocyte cells are stimulated to grow by injecting the antigen in association with an adjuvant such as complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis). At some time (e.g. , at least two weeks) after the first injection, mice are boosted by injection with a second dose of the antigen mixed with incomplete Freund's adjuvant.
A few weeks after the second injection, mice are tail bled and the sera titered by immunoprecipitation against radiolabeled antigen. The process of boosting and titering can be repeated until a suitable titer is achieved. The spleen of the mouse with the highest titer is removed and the spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
Mutant lymphocyte cells known as myeloma cells are obtained from laboratory animals in which such cells have been induced to grow by a variety of well-known methods. Myeloma cells lack the salvage pathway of nucleotide biosynthesis. Because myeloma cells are tumor cells, they can be propagated indefinitely in tissue culture, and are thus "immortal". Numerous cultured cell lines of myeloma cells from mice and rats, such as murine NS-1 myeloma cells, have been established.
Myeloma cells are combined under conditions appropriate to foster fusion with the normal antibody-producing cells from the spleen of the mouse or rat injected with the antigen/polypeptide of the presently disclosed subject matter. Fusion conditions include, for example, the presence of polyethylene glycol. The resulting fused cells are hybridoma cells. Like myeloma cells, hybridoma cells grow indefinitely in culture. Hybridoma cells are separated from unfused myeloma cells by culturing in a selection medium such as HAT media (hypoxanthine, aminopterin, and thymidine). Unfused myeloma cells lack the enzymes necessary to synthesize nucleotides from the salvage pathway because they are killed in the presence of aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not continue to grow in tissue culture. Thus, only cells that have successfully fused (hybridoma cells) can grow in the selection media.
Each of the surviving hybridoma cells produces a single antibody. These cells are then screened for the production of the specific antibody immunoreactive with an antigen/polypeptide of the presently disclosed subject matter. Single cell hybridomas are isolated by limiting dilutions of the hybridomas. The hybridomas are serially diluted many times and, after the dilutions are allowed to grow, the supernatant is tested for the presence of the monoclonal antibody. The clones producing that antibody are then cultured in large amounts to produce an antibody of the presently disclosed subject matter in convenient quantity.
By use of a monoclonal antibody of the presently disclosed subject matter, specific polypeptides and polynucleotide of the presently disclosed subject matter can be recognized as antigens, and thus identified. Once identified, those polypeptides and polynucleotide can be isolated and purified by techniques such as antibody-affinity chromatography. In antibody-affinity chromatography, a monoclonal antibody is bound to a solid substrate and exposed to a solution containing the desired antigen. The antigen is removed from the solution through an immunospecific reaction with the bound antibody. The polypeptide or polynucleotide is then easily removed from the substrate and purified.
II. G. Detecting a Polynucleotide or a Polypeptide Alternatively, the presently disclosed subject matter provides a process of detecting a polypeptide of the presently disclosed subject matter, wherein the process comprises immunoreacting the polypeptides with antibodies prepared according to the process described above to form antibody- polypeptide conjugates, and detecting the conjugates. In yet another embodiment, the presently disclosed subject matter provides a process of detecting messenger RNA transcripts that encode a polypeptide of the presently disclosed subject matter, wherein the process comprises hybridizing the messenger RNA transcripts with polynucleotide sequences that encode the polypeptide to form duplexes; and detecting the duplex. Alternatively, the presently disclosed subject matter provides a process of detecting DNA molecules that encode a polypeptide of the presently disclosed subject matter, wherein the process comprises hybridizing DNA molecules with a polynucleotide that encodes that polypeptide to form duplexes; and detecting the duplexes.
The detection and screening assays disclosed herein can optionally be used as a prognosis tool and/or diagnostic aid. MOR-1 K encoding polypeptides and nucleic acids can be readily used in clinical setting as a prognostic and/or diagnostic indicator for screening for levels of expression of 6TM mu-opioid receptor isoforms, or alterations in native sequences.
The presently disclosed subject matter provides a process of screening a biological sample for the presence of a 6TM mu-opioid receptor isoform polypeptide. A biological sample to be screened can be a biological fluid such as extracellular or intracellular fluid, or a cell or tissue extract or homogenate. A biological sample can also be an isolated cell (e.g., in culture) or a collection of cells such as in a tissue sample or histology sample. A tissue sample can be suspended in a liquid medium or fixed onto a solid support such as a microscope slide. In accordance with a screening assay process, a biological sample is exposed to an antibody immunoreactive with the polypeptide whose presence is being assayed. Typically, exposure is accomplished by forming an admixture in a liquid medium that contains both the antibody and the candidate polypeptide. Either the antibody or the sample with the polypeptide can be affixed to a solid support (e.g., a column or a microtiter plate). Additional details of methods for such assays are known in the art. The presence of polypeptide in the sample is detected by evaluating the formation and presence of antibody-polypeptide conjugates. Techniques for detecting such antibody- antigen conjugates or complexes are well known in the art and include but are not limited to centrifugation, affinity chromatography and the like, and binding of a secondary antibody to the antibody-candidate receptor complex.
In one embodiment, detection is accomplished by detecting an indicator affixed to the antibody. Exemplary and well-known indicators include radioactive labels (e.g., 32P, 1251, 14C), a second antibody or an enzyme such as horseradish peroxidase. Techniques for affixing indicators to antibodies are known in the art.
In another aspect, the presently disclosed subject matter provides a process of screening a biological sample for the presence of antibodies immunoreactive with a 6TM mu-opioid receptor polypeptide.
A DNA or RNA molecule and particularly a DNA segment or polynucleotide can be used for hybridization to a DNA or RNA source or sample suspected of encoding a 6TM mu-opioid receptor; such molecules are referred to as "probes," and such hybridization is "probing". Such probes can be made synthetically. The probing is usually accomplished by hybridizing the oligonucleotide to a DNA source suspected of possessing a MOR-1 K gene product. In some cases, the probes constitute only a single probe, and in others, the probes constitute a collection of probes based on a certain amino acid sequence or sequences of the polypeptide and account in their diversity for the redundancy inherent in the genetic code.
Other molecules which are neither DNA nor RNA but are capable of hybridizing in a similar manner and which are designed structurally to mimic the DNA or RNA sequence of a MOR-1 K gene product are also provided. Here, a suitable source to examine is capable of expressing a polypeptide of the presently disclosed subject matter and can be a genomic library of a cell line of interest. Alternatively, a source of DNA or RNA can include total DNA or RNA from the cell line of interest. Once the hybridization process has identified a candidate DNA segment, a positive clone can be confirmed by further hybridization, restriction enzyme mapping, sequencing and/or expression and testing.
Alternatively, such DNA molecules can be used in a number of techniques including their use as: (1) diagnostic tools to detect sequences in DNA derived from patient's cells; (2) reagents for detecting and isolating other members of the polypeptide family and related polypeptides from a DNA library potentially containing such sequences; and (3) primers for hybridizing to related sequences for the purpose of amplifying those sequences.
As set forth above, in certain aspects, DNA sequence information provided by the presently disclosed subject matter allows for the preparation of probes that specifically hybridize to encoding sequences of a selected MOR-1 K gene product. In these aspects, probes of an appropriate length are prepared based on a consideration of the encoding sequence for a polypeptide of the presently disclosed subject matter. The ability of such probes to specifically hybridize to other encoding sequences lends them particular utility in a variety of embodiments. Most importantly, the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
"Primers" of the presently disclosed subject matter are designed to be "substantially" complementary to each strand of the genomic locus to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' sequences flanking the transition to hybridize therewith and permit amplification of the genomic locus.
Oligonucleotide primers of the presently disclosed subject matter are employed in the amplification method that is an enzymatic chain reaction that produces exponential quantities of polymorphic locus relative to the number of reaction steps involved. Typically, one primer is complementary to the negative (-) strand of the polymorphic locus and the other is complementary to the positive (+) strand. Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA polymerase I (Klenow) and nucleotides, results in newly synthesized + and - strands containing the target polymorphic locus sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target polymorphic locus sequence) defined by the primers. The product of the chain reaction is a discreet nucleic acid duplex with termini corresponding to the ends of the specific primers employed.
The oligonucleotide primers of the presently disclosed subject matter can be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidites are used as starting materials and can be synthesized as described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1862. One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
Any nucleic acid specimen, in purified or non-purified form, can be utilized as the starting nucleic acid or acids, providing it contains, or is suspected of containing, a nucleic acid sequence containing the polymorphic locus. Thus, the method can amplify, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA can be single stranded or double stranded. In the event that RNA is to be used as a template, enzymes, and/or conditions optimal for reverse transcribing the template to DNA would be utilized. In addition, a DNA-RNA hybrid that contains one strand of each can be utilized. A mixture of nucleic acids can also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers can be so utilized. The specific nucleic acid sequence to be amplified, i.e., the polymorphic locus, can be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified is present initially in a pure form; it can be a minor fraction of a complex mixture, such as contained in whole human DNA.
DNA utilized herein can be extracted from a body sample, such as blood, tissue material (e.g., brain or kidney tissue), and the like by a variety of techniques such as that described by Maniatis et. al. (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, New York. If the extracted sample is impure, it can be treated before amplification with an amount of a reagent effective to open the cells, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily.
The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90-100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization"), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization can also be added together with the other reagents if it is heat stable. This synthesis (or amplification) reaction can occur at room temperature up to a temperature above which the agent for polymerization no longer functions. Thus, for example, if DNA polymerase is used as the agent, the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature.
The agent for polymerization can be any compound or system that will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase, polymerase muteins, reverse transcriptase, other enzymes, including heat-stable enzymes {i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation), such as Taq polymerase. Suitable enzyme will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each polymorphic locus nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths.
The newly synthesized strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described herein and this hybrid is used in subsequent steps of the method. In the next step, the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules.
The steps of denaturing, annealing, and extension product synthesis can be repeated as often as needed to amplify the target polymorphic locus nucleic acid sequence to the extent necessary for detection. The amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion. See McPherson et al., eds. (1991) PCR. A Practical Approach, IRL Press, Oxford University Press, New York, New York. The amplification products can be detected by Southern blot analysis with or without using radioactive probes. In one such method, for example, a small sample of DNA containing a very low level of the nucleic acid sequence of the polymorphic locus is amplified, and analyzed via a Southern blotting technique or similarly, using dot blot analysis. The use of non-radioactive probes or labels is facilitated by the high level of the amplified signal. Alternatively, probes used to detect the amplified products can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.
Sequences amplified by the methods of the presently disclosed subject matter can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as dideoxy sequencing, PCR, oligomer restriction (Saiki et al. (1985) Bio/Technology 3:1008-1012), allele-specific oligonucleotide (ASO) probe analysis (Conner et ah (1983) Proc Natl Acad Sci USA 80:278), oligonucleotide ligation assays (OLAs) (Landgren et al. (1988) Science 241 :1007), and the like. Molecular techniques for DNA analysis have been reviewed (Landqren et al. (1988) Science 242:229-237).
Preferably, the method of amplifying is by PCR, as described herein and in U.S. Patent Nos. 4,683,195; 4,683,202; and 4,965,188 each of which is hereby incorporated by reference; and as is commonly used by those of ordinary skill in the art. Alternative methods of amplification have been described and can also be employed as long as a MOR-1 locus amplified by PCR using primers of the presently disclosed subject matter is similarly amplified by the alternative approach. Such alternative amplification systems include but are not limited to self-sustained sequence replication, which begins with a short sequence of RNA of interest and a T7 promoter. Reverse transcriptase transcribes the RNA into cDNA and degrades the RNA, followed by reverse transcriptase polymerizing a second strand of DNA. Another nucleic acid amplification technique is nucleic acid sequence-based amplification (NASBA™) which uses reverse transcription and T7 RNA polymerase and incorporates two primers to target its cycling scheme. NASBA™ amplification can begin with either DNA or RNA and finish with either, and amplifies to about 108 copies within 60 to 90 minutes. Alternatively, nucleic acid can be amplified by ligation-activated transcription (LAT). LAT works from a single-stranded template with a single primer that is partially single-stranded and partially double-stranded. Amplification is initiated by ligating a cDNA to the promoter olignucleotide and within a few hours, amplification is about 108 to about 109 fold. The QB replicase system can be utilized by attaching an RNA sequence called MDV-1 to RNA complementary to a DNA sequence of interest. Upon mixing with a sample, the hybrid RNA finds its complement among the specimen's mRNAs and binds, activating the replicase to copy the tag-along sequence of interest.
Another nucleic acid amplification technique, ligase chain reaction (LCR), works by using two differently labeled halves of a sequence of interest that are covalently bonded by ligase in the presence of the contiguous sequence in a sample, forming a new target. The repair chain reaction (RCR) nucleic acid amplification technique uses two complementary and target-specific oligonucleotide probe pairs, thermostable polymerase and ligase, and DNA nucleotides to geometrically amplify targeted sequences. A 2-base gap separates the oligo probe pairs, and the RCR fills and joins the gap, mimicking normal DNA repair.
Nucleic acid amplification by strand displacement activation (SDA) utilizes a short primer containing a recognition site for Hinc Il with short overhang on the 5' end which binds to target DNA. A DNA polymerase fills in the part of the primer opposite the overhang with sulfur-containing adenine analogs. Hinc Il is added but only cuts the unmodified DNA strand. A DNA polymerase that lacks 5' exonuclease activity enters at the site of the nick and begins to polymerize, displacing the initial primer strand downstream and building a new one which serves as more primer.
SDA produces greater than about a 107-fold amplification in 2 hours at 37°C. Unlike PCR and LCR, SDA does not require instrumented temperature cycling. Another amplification system useful in the method of the presently disclosed subject matter is the QB Replicase System. Although PCR is the preferred method of amplification if the presently disclosed subject matter, these other methods can also be used. Thus, the term "amplification technique" as used herein and in the claims is meant to encompass all the foregoing methods.
In another embodiment of the presently disclosed subject matter a method is provided for identifying a subject having a polymorphism of a MOR-1 gene, comprising sequencing a target nucleic acid of a sample from a subject by dideoxy sequencing, preferably following amplification of the target nucleic acid.
In another embodiment of the presently disclosed subject matter a method is provided for identifying a subject having a polymorphism of a MOR-1 gene, comprising contacting a target nucleic acid of a sample from a subject with a reagent that detects the presence of a MOR-1 polymorphism and detecting the reagent. A number of hybridization methods are well known to those skilled in the art.
Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those of ordinary skill in the art. Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1 ,00OmM, typically less than 50OmM, and preferably less than 20OmM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wethmur & Davidson (1986) J MoI Biol 31.349-370. Accordingly, a nucleotide sequence of the presently disclosed subject matter can be used for its ability to selectively form duplex molecules with complementary stretches of a OPRM1 gene or a MOR-1 K gene product. Depending on the application envisioned, one employs varying conditions of hybridization to achieve varying degrees of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, one typically employs relatively stringent conditions to form the hybrids. For example, one selects relatively low salt and/or high temperature conditions, such as provided by 0.02M-0.15M salt at temperatures of about 50°C to about 70°C including particularly temperatures of about 55°C, about 600C and about 65°C. Such conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate polypeptide coding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions are typically needed to allow formation of the heteroduplex. Under such circumstances, one employs conditions such as 0.15M-0.9M salt, at temperatures ranging from about 200C to about 55°C, including particularly temperatures of about 25°C, about 37°C, about 45°C, and about 500C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it is advantageous to employ a nucleic acid sequence of the presently disclosed subject matter in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator reagents are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one likely employs an enzyme tag such a urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates are known which can be employed to provide a reagent visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein are useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the sample containing test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions depend inter alia on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, efc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
The materials for use in the method of the presently disclosed subject matter are ideally suited for the preparation of a screening kit. Such a kit can comprise a carrier having compartments to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method. For example, one of the containers can comprise an amplifying reagent for amplifying a MOR-1 or MOR-1 K DNA, such as the necessary enzyme(s) and oligonucleotide primers for amplifying target DNA from the subject.
In another aspect, the presently disclosed subject matter provides assay kits for detecting the presence of a polypeptide of the presently disclosed subject matter in biological samples, where the kits comprise a first antibody capable of immunoreacting with the polypeptide. The assay kits of the presently disclosed subject matter can further comprise a second container containing a second antibody that immunoreacts with the first antibody. The antibodies used in the assay kits of the presently disclosed subject matter can be monoclonal antibodies. The first antibody can be affixed to a solid support. The first and second antibodies can comprise an indicator, such as but not limited to a radioactive label or an enzyme.
The presently disclosed subject matter also provides an assay kit for screening agents. Such a kit can contain a polypeptide of the presently disclosed subject matter. The kit can additionally contain reagents for detecting an interaction between an agent and a polypeptide of the presently disclosed subject matter.
In an alternative aspect, the presently disclosed subject matter provides assay kits for detecting the presence, in biological samples, of a polynucleotide that encodes a polypeptide of the presently disclosed subject matter, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or more contiguous nucleotide bases of, as an example, any of SEQ ID NOs: 1 or 2.
In some embodiments, one or more PCR probes can be used that hybridize specifically to one or more exons present in an MOR-1 K gene product. For examples, one or more probes selected from SEQ ID NOS: 5-9 can be used to detect a gene product comprising exon 13 of the human mu- opioid receptor gene. A probe selected from SEQ ID NOS: 5-9 can be used in combination with a probe specific for another exon present in the MOR-1 K gene products. Thus, the probe of SEQ ID NOS: 5-9 can be used in combination with a probe of SEQ ID NOS: 10-17, which are each specific for one of exons 2, 4, 5, or 0/7 of the human mu-opioid receptor gene. In some embodiments of the presently disclosed subject matter, two pairs of isolated oligonucleotide primers are provided. These sets of primers are optionally derived from a MOR-1 exon. The oligonucleotide primers are useful, for example, in detecting a polymorphism. The primers direct amplification of a target polynucleotide prior to sequencing. In another embodiment of the presently disclosed subject matter isolated allele specific oligonucleotides (ASO) are provided. The allele specific oligonucleotides are also useful in detecting a polymorphism. In some embodiments, a probe can be selected for its ability to hybridize to a 5'-reguIatory region, such as a 5' promoter region for exon 13 of MOR-1 K.
In some embodiments, the probe is selective for the 5'UTR of a MOR-1 K gene product. In some embodiments, the probe is selective for an allelic variant of an internal ribosome binding site (IRES) in the 5'UTR of the MOR-1 K.
In another embodiment, the presently disclosed subject matter provides assay kits for detecting the presence, in a biological sample, of antibodies immunoreactive with a polypeptide of the presently disclosed subject matter, the kits comprising a MOR-1 K polypeptide that immunoreacts with the antibodies.
HL Methods of Screening Compounds for the Ability to Modulate 6TM Mu- Opioid Receptor Activity 6TM mu-opioid receptor isoform modulators can be identified by: providing a test sample comprising a 6TM mu-opioid receptor isoform polypeptide; administering a test molecule to the test sample; and determining the effect of the test molecule on the activity of the polypeptide. A test molecule can be any molecule having any chemical structure. For example, a test molecule can be a polypeptide, carbohydrate, lipid, amino acid, nucleic acid, fatty acid, or steroid. In addition, a test molecule can be lipophilic, hydrophilic, plasma membrane permeable, or plasma membrane impermeable.
The presently disclosed subject matter provides several assays that can be used to identify 6TM mu-opioid receptor modulators. Such assays involve monitoring at least one of the biological responses mediated by a 6TM mu- opioid receptor in cells expressing a polypeptide having 6TM mu-opioid receptor activity such as cells containing an exogenous nucleic acid molecule that expresses a polypeptide having 6TM mu-opioid receptor activity. As described herein, 6TM mu-opioid receptor-mediated responses include, without limitation, changes in intracellular calcium concentration, cAMP levels, nitric oxide release, and coupling to Ga3 protein. Thus, a 6TM mu-opioid receptor agonist can be identified using an assay that monitors intracellular calcium concentration, cAMP levels, nitric oxide release, coupling to Gαs protein or an combination of these measures in cells transfected with a nucleic acid molecule that expresses a polypeptide having 6TM mu-opioid receptor activity. Furthermore, in some embodiments, the test sample is a non-human animal and determining the effect of the test compound is estimated by assessment of pain-related behavior in the animal. Intracellular calcium concentrations can be monitored using any method.
For example, intracellular calcium concentrations can be monitored using a dye that detects calcium ions. In this case, cells can be loaded with a fluorescent dye (e.g., fura-2) and monitored by dual emission microfluorimetry. The fura-2 loading process can involve washing the cells (e.g., one to four times) with incubation medium lacking calcium. This medium can be balanced with sucrose to maintain osmolarity. After washing, the cells can be incubated (e.g., 30 minutes) with loading solution. This loading solution can contain, for example, 5 μM fura-2/AM and a non-ionic/non-denaturing detergent such as Pluronic F- 127. The non-ionic/non-denaturing detergent can help disperse the acetoxymethyl (AM) esters of fura-2. After incubation with the loading solution, the cells can be washed (e.g., one to four times) with, for example, PBS without calcium or magnesium to remove extracellular dye.
Once loaded, the intracellular calcium concentration can be calculated from the fluorescence ratio (340 and 380 nm excitation and 510 nm emission wavelength) according to the following equation: [Ca2+]J= (R-Rmjπ) k dβ/(Rmax - R); where R=fluorescence ratio recorded from the cell; Rmjn = fluorescence ratio of fura-2 free acid recorded in absence of Ca 2+; Rmaχ= fluorescence ratio of fura-2 free acid recorded in saturating concentration of Ca2+; kd = calcium dissociation constant of the dye; and β = the ratio of fluorescence of fura-2 free acid in the Ca2+ free form to the Ca2+ saturated form recorded at the wavelength used in the denominator of the ratio. Using an image processing system such as a COMPIX C-640 SIMCA (Compix Inc., Mars, Pennsylvania, United States of America) system with an inverted microscope, images can be acquired for analysis every 0.4 seconds. Nitric oxide (NO) release can be monitored directly or indirectly using any method. For example, a NO-specific amperometric probe can be used to measure directly the NO released from cultured cells or tissue fragments as described elsewhere (see Stefano, et al., J. Biol. Chem., 270, 30290 (1995); and Magazine, et al., J. Immunol., 156, 4845 (1996)). Using this NO-specific probe, the concentration of NO gas in solution can be measured in real-time with, for example, a DUO 18 computer data acquisition system obtained from World Precision Instruments. Briefly, the cells or tissue fragments can be placed in a superfusion chamber containing, for example, 2 ml_ PBS. In addition, a micromanipulator (e.g., a micromanipulator obtained from Zeiss- Eppendorff) can be attached to the stage of an inverted microscope to aid in positioning the amperometric probe 15 μm above the surface of a cell or tissue fragment. Prior to obtaining measurements, the amperometric probe can be calibrated by generating a standard curve using different concentrations of a nitrosothiol donor such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) obtained from Sigma (St. Louis, Missouri, United States of America). In addition, the amperometric probe can be equilibrated in the same solution (e.g., PBS) used to incubate the cells or tissue fragments during analysis. The coupling of the 6TM isoform of OPRM 1 with the Gαs isoform of the
Ga protein can be monitored directly or indirectly using any method. For example, for immunoprecipitation the cells can be lysed using radioimmunoprecipitation assay (RIPA) buffer and centrifuged at maximum RPM for 20 minutes. Supernatants should be collected and used for Co- immunoprecipitation (Co-Ip) experiments. Following overnight incubation on a rotary shaker at 4°C with anti-FLAG (MOR1 K) or anti-MYC (MOR1 ) antibodies, Ip beads (Pierce Biotechnology) are added and the samples incubated for 6 hours at 4°C. The samples were then centrifuged at 14000 rpm for 10 min, the supernatants should be discarded and the beads re-suspended in 50% RIPA/PBS buffer. The procedure can be repeated 3 times. The samples can then be boiled and run on 12% SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare Bio-Sciences). After blocking (overnight at 4°C) with 5% non-fat dried milk in Tris-buffered saline with Tween 20 (blocking buffer, TBS-T, 150 mM NaCI, 20 mM Tris-HCI, pH 7.5, 0.1 % Tween 20), the membranes can be probed with primary antibodies [anti- Gots (K-20): sc-823, anti-Gd (C-10): sc-262 and anti-Gβ (T-20): sc-378; Santa Cruz Biotechnology, 1 :1 ,000] overnight at 4°C . After washing in TBS-T (three times, 5 min each), the blots can be incubated for 4 hours at room temperature with a horseradish peroxidase-conjugated secondary antibody (1 :15,000; GE Healthcare Bio-Sciences). The antibody-antigen complexes can be detected using the ECL system (Amersham) and visualized with photosensitive film (Kodak). In accordance with the presently disclosed subject matter invention there are also provided methods for screening candidate compounds for the ability to modulate in vivo six transmembrane μ-opioid receptor isoform levels and/or activity. Representative modulators of six transmembrane μ-opioid receptor isoform levels can comprise modulators of transcription or expression. Compositions that increase or decrease the transcription or expression of six transmembrane μ-opioid receptor isoform-encoding genes have clinical application for the modulation of the biological activity of six transmembrane μ- opioid receptor isoforms.
Thus provided herein is a method for discovery of compounds that modulate the expression levels of six transmembrane μ-opioid receptor isoform-encoding genes. The general approach is to screen compound libraries for substances which increase or decrease expression of six transmembrane μ- opioid receptor isoform-encoding genes. Exemplary techniques are described in U.S. Patent Nos. 5,846,720 and 5,580,722, the entire contents of each of which are herein incorporated by reference.
While the following terms are believed to be well understood by one of skill in the art, the following definitions are set forth to facilitate explanation of the invention.
"Transcription" means a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene. The process includes, but is not limited to the following steps: (a) the transcription initiation, (b) transcript elongation, (c) transcript splicing, (d) transcript capping, (e) transcript termination, (f) transcript polyadenylation, (g) nuclear export of the transcript, (h) transcript editing, and (i) stabilizing the transcript. "Expression" generally refers to the cellular processes by which a biologically active polypeptide is produced from RNA. "Transcription factor" means a cytoplasmic or nuclear protein which binds to such gene, or binds to an RNA transcript of such gene, or binds to another protein which binds to such gene or such RNA transcript or another protein which in turn binds to such gene or such RNA transcript, so as to thereby modulate expression of the gene. Such modulation can additionally be achieved by other mechanisms; the essence of "transcription factor for a gene" is that the level of transcription of the gene is altered in some way.
In accordance with the presently disclosed subject matter there is provided a method of identifying a candidate compound or molecule that is capable of modulating the transcription level of a gene encoding a six transmembrane μ-opioid receptor isoform and thus is capable of acting in the modulation of six transmembrane μ-opioid receptor isoform effects. Such modulation can be direct, i.e., through binding of a candidate molecule directly to the nucleotide sequence, whether DNA or RNA transcript, or such modulation can be achieved via one or more intermediaries, such as proteins other than six transmembrane μ-opioid receptor isoform which are affected by the candidate compound and ultimately modulate six transmembrane μ-opioid receptor isoform transcription by any mechanism, including direct binding, phosphorylation or dephosphorylation, efc. This method comprises contacting a cell or nucleic acid sample with a candidate compound or molecule to be tested. These samples contain nucleic acids which can contain elements that modulate transcription and/or translation of a six transmembrane μ-opioid receptor isoform gene, such as a promoter or putative upstream regulatory region (representative of such as disclosed herein), and a DNA sequence encoding a polypeptide which can be detected in some way. Thus, the polypeptide can be described as a "reporter" or "marker."
Optionally, the candidate compound directly and specifically transcriptionally modulates expression of the six transmembrane μ-opioid receptor isoform- encoding gene. The DNA sequence is coupled to and under the control of the promoter, under conditions such that the candidate compound or molecule, if capable of acting as a transcriptional modulator of the gene encoding six transmembrane μ-opioid receptor isoform, causes the polypeptide to be expressed and so produces a detectable signal, which can be assayed quantitatively and compared to an appropriate control. Candidate compounds or molecules of interest can include those which increase or decrease, i.e., modulate, transcription from the regulatory region. The reporter gene can encode a reporter known in the art, such as luciferase, or it can encode six transmembrane μ-opioid receptor isoform.
In certain embodiments of the presently disclosed subject matter the polypeptide so produced is capable of complexing with an antibody or is capable of complexing with biotin. In this case the resulting complexes can be detected by methods known in the art. The detectable signal of this assay can also be provided by messenger RNA produced by transcription of said reporter gene. Exactly how the signal is produced and detected can vary and is not the subject of the presently disclosed subject matter; rather, the presently disclosed subject matter provides the nucleotide sequences and/or putative regulatory regions of six transmembrane μ-opioid receptor isoform for use in such an assay. The molecule to be tested in these methods can be a purified molecule, a homogenous sample, or a mixture of molecules or compounds. Further, in representative embodiments, the DNA in the cell comprises more than one modulatable transcriptional regulatory sequence. In accordance with the presently disclosed subject matter there is also provided a rapid and high throughput screening method that relies on the methods described above. This screening method comprises separately contacting each of a plurality of substantially identical samples. In such a screening method the plurality of samples preferably comprises more than about 104 samples, or more preferably comprises more than about 5 x 104 samples. SEQ ID NO: 3
MKTATNIYIFNLALADALXTSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSXF TLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGS IDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKD RNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSC LNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLE NLEAETAPLP* where as SCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQ
LENLEAETAPLP* is intra-cellular C-terminal domain and can be substituted with different polypeptide
SEQ ID NO: 4
MDSSAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSL CPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALAD ALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHP VKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWEN LLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAV FIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRC FREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLENLEAETAPLP* where as
MDSSAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSL
CPPTGSPS is extra-cellular N-terminal domain and can be substituted with different polypeptide and SCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQ
LENLEAETAPLP* is intra-cellular C-terminal domain and can be substituted with different polypeptide
It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:
1. An isolated nucleic acid sequence consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 1 or a nucleic acid sequence having at least a 98% sequence homology to SEQ ID NO: 1 over its entire length.
2. An isolated nucleic acid sequence consisting essentially of a nucleic acid sequence selected from SEQ ID NO: 2 or a sequence having at least a 98% sequence homology to SEQ ID NO: 2 over its entire length.
3. An isolated nucleic acid sequence coding for a protein consisting essentially of an amino acid sequence of SEQ ID NO: 3.
4. An isolated nucleic acid sequence coding for a protein comprising amino acids 1-300 of SEQ ID NO: 3.
5. A nucleic acid sequence that is at least 97% identical to a contiguous 902 nucleotide sequence selected from nucleotides 1309-2211 of SEQ ID NO: 1 or nucleotides 1290-2192 of SEQ ID NO: 2.
6. A recombinant vector comprising the nucleic acid sequence of one of claims 1-4.
7. A recombinant host cell comprising the nucleic acid sequence of one of claims 1-4.
8. A substantially pure polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 3 or a sequence having a 97% or greater sequence homology to SEQ ID NO: 3 over its entire length.
9. A substantially pure polypeptide consisting essentially of amino acids 1-300 of SEQ ID NO: 3 or a sequence having at least a 97% sequence homology to the sequence comprising amino acids 1-300 of SEQ ID NO: 3 over its entire length.
10. A recombinant cell expressing the polypeptide of claim 8 or claim 9.
11. A method of screening candidate substances for an ability to modulate activity of a six-transmembrane μ-opioid receptor isoform, the method comprising: providing a test sample comprising a polypeptide of claim 8 or 9; administering a test molecule to the test sample; and determining the effect of the test molecule on the activity of the polypeptide.
12. The method of claim 11 , wherein the test molecule is selected from the group consisting of a polypeptide, a nucleic acid oligonucleotide, an exogenous vector coding for a nucleic acid oligonucleotide or polypeptide, a carbohydrate, a lipid, an amino acid, a fatty acid, a steroid, and a low molecular weight organic molecule.
13. The method of claim 11 , wherein determining the effect of the test molecule on the activity of the polypeptide comprises measuring a first activity level of the polypeptide prior to administering the test molecule to the test sample, measuring a second activity level of the polypeptide after administering the test molecule to the test sample, and comparing the first and second activity levels.
14. The method of claim 11 , further comprising determining the effect of the test molecule on the activity of a polypeptide comprising the sequence of SEQ ID NO: 4 or amino acids 67-330 of SEQ ID NO: 4 and comparing said effect with the effect of the test molecule on the activity of the polypeptide of claim 8 or 9.
15. The method of claim 11 , wherein the test sample is a cell.
16. The method of claim 15 wherein the polypeptide is provided to the cell from an exogenous source.
17. The method of claim 15, wherein the cell expresses the polypeptide.
18. The method of claim 17, wherein the cell is a recombinant cell.
19. The method of claim 11 , wherein determining the effect of the test molecule comprises measuring an amount or a change in the amount of one or more of the group consisting of cAMP, calcium, nitric oxide or coupling to Ga5 protein in the test sample.
20. The method of claim 11 , wherein the test sample is a non-human animal.
21. The method of claim 20, wherein determining the effect of the test compound is estimated by assessment of pain-related behavior in the animal.
22. The method of claim 20, wherein the animal is a genetically modified animal.
23. The method of claim 22, wherein the animal possesses modulated six transmembrane μ-opioid receptor isoform activity, modulated seven transmembrane μ-opioid receptor isoform activity, or a combination thereof.
24. The method of claim 23, wherein the animal is a transgenic animal that overexpresses a polypeptide of claim 8 or 9.
25. The method of claim 23, wherein the animal is a knock-out animal that under expresses a seven transmembrane μ-opioid receptor isoform.
26. The method of claim 25, wherein the seven transmembrane μ- opioid receptor isoform is a polypeptide comprising the sequence of SEQ ID
NO: 4 or amino acids 67-330 of SEQ ID NO: 4.
27. A transgenic non-human animal, wherein the transgenic non- human animal overexpresses a six-transmembrane μ-opioid receptor isoform comprising a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology to amino acids 1-300 of SEQ ID NO: 3 over the entire length of the amino acid sequence.
28. A method for identifying an antagonist or an agonist specific for a six transmembrane μ-opioid receptor isoform, wherein the isoform comprises a polypeptide consisting essentially of SEQ ID NO: 3, amino acids 1-300 of SEQ ID NO: 3, or an amino acid sequence having at least a 97% homology thereto over the entire length of the amino acid, the method comprising: contacting a cell with a test molecule and a μ-opioid receptor agonist or antagonist, wherein said cell comprises a nucleic acid molecule consisting essentially of one of SEQ ID NOs: 1 or 2 or a sequence having at least a 98% homology thereto, and wherein said cell expresses said polypeptide; and determining whether or not said test molecule reduces or prevents a non-analgesic or hyperanalgesic μ-opioid receptor-mediated response induced by said μ-opioid receptor agonist or antagonist.
29. A method of producing an antibody immunoreactive with a six transmembrane μ-opioid receptor isoform polypeptide, the method comprising steps of: transfecting a recombinant host cell with a nucleic acid molecule of any of claims 1-4 which encodes a six transmembrane μ-opioid receptor isoform polypeptide; culturing the host ceil under conditions sufficient for expression of the polypeptide; recovering the polypeptide; and preparing an antibody to the polypeptide.
30. The method of claim 29, wherein the nucleic acid molecule consists of SEQ ID NO: 1 or SEQ ID NO: 2.
31. A method of detecting a six transmembrane μ-opioid receptor polypeptide, the method comprising immunoreacting the polypeptide with an antibody prepared according to the method of claim 29 to form an antibody- polypeptide conjugate; and detecting the conjugate.
32. A method of detecting a nucleic acid molecule that encodes a six transmembrane μ-opioid receptor isoform polypeptide in a biological sample containing nucleic acid material, the method comprising the steps of: hybridizing the nucleic acid molecule having a sequence complementary to one of SEQ ID NO: 1 or SEQ ID NO: 2 under stringent hybridization conditions to the nucleic acid material of the biological sample, thereby forming a hybridization duplex; and detecting the hybridization duplex.
33. An assay kit for detecting the presence of a six transmembrane μ- opioid receptor isoform in a biological sample, the kit comprising a first container comprising a first antibody capable of immunoreacting with a polypeptide of claim 8 or claim 9.
34. The assay kit of claim 33, further comprising a second container containing a second antibody that immunoreacts with the first antibody.
35. The assay kit of claim 34, wherein the first antibody and the second antibody comprise monoclonal antibodies.
36. The assay kit of claim 33, wherein the first antibody is affixed to a solid support.
37. The assay kit of claim 34, wherein the first and second antibodies each comprise an indicator.
38. The assay kit of claim 37, wherein the indicator is a radioactive label or an enzyme.
39. An assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a six transmembrane μ-opioid receptor isoform polypeptide, the kit comprising a polypeptide of claim 8 or claim 9 that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
40. An assay kit for detecting the presence, in biological samples, of a six transmembrane μ-opioid receptor isoform polypeptide, the kit comprising a first container that contains a nucleic acid molecule identical or complementary to a segment of at least ten contiguous nucleotide bases of the nucleic acid molecule of any of claims 1-4.
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