Identification and characterization of multiple splice variants of the mu-opioid receptor gene

Abstract

The present invention encompasses novel splice variant forms of the mu-opioid receptor-1 (MOR-1) and the polynucleotide sequences encoding the MOR-1 splice variants. The invention further encompasses methods of screening for compositions regulating the MOR-1 splice variant activities and the development of therapeutic modalities directed to regulating activity. Regulation of the MOR-1 splice variant activities may impact the physiologic processes of analgesia and weight management.

1 . An isolated MOR-1C splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         2 . An isolated MOR-1D splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         3 . An isolated MOR-1E splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         4 . An isolated MOR-1F splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         5 . An isolated MOR-1G splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         6 . An isolated MOR-1A splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         7 . An isolated MOR-1H splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         8 . An isolated MOR-1B II splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         9 . An isolated MOR-1B I splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         10 . An isolated MOR-1I splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         11 . An isolated MOR-1J splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         12 . An isolated human MOR-1 splice variant polypeptide that consists essentially of the amino acid residues depicted in FIG. 3.  
     
     
         13 . The polypeptide as in one of the preceding claims in which the polypeptide comprises a heterodimeric or homodimeric composition.  
     
     
         14 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1c as depicted in FIG. 2.  
     
     
         15 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1d as depicted in FIG. 2.  
     
     
         16 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1e as depicted in FIG. 2.  
     
     
         17 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1f as depicted in FIG. 2.  
     
     
         18 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1b II as depicted in FIG. 2.  
     
     
         19 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1a as depicted in FIG. 2.  
     
     
         20 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1b I as depicted in FIG. 2.  
     
     
         21 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1i as depicted in FIG. 2.  
     
     
         22 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1j as depicted in FIG. 2.  
     
     
         23 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1g as depicted in FIG. 2.  
     
     
         24 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of MOR-1h as depicted in FIG. 2.  
     
     
         25 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of human MOR-1 as depicted in FIG. 2.  
     
     
         26 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of 2730510 as depicted in FIG. 2.  
     
     
         27 . An isolated polynucleotide, or a complementary strand thereto that hybridizes under stringent conditions, wherein the nucleotide fragment consists essentially of 161416 as depicted in FIG. 2.  
     
     
         28 . A method of screening compositions for opioid activity comprising the steps of a) obtaining a control cell that does not express an MOR-1 splice variant polypeptide; b) obtaining a test cell that is the same as the control cell except that it expresses an MOR-1 splice variant polypeptide as in any one of claims  122 - 133 ; c)contacting the control cell and test cell with an amount of an opioid sufficient to exert a physiologic effect; d) separately measuring the physiologic effect of the composition on the control cell and test cell; and e) comparing the physiologic effect of the composition to the physiologic effect of the opioid, where determination of a physiologic effect of the composition is expressed relative to that of the opioid.  
     
     
         29 . The method according to claim  149 , where the composition is selected from the group consisting of synthetic combinatorial libraries of small molecule ligands, eukaryotic whole cell lysates or extracts, or media conditioned by cultured eukaryotic cells.  
     
     
         30 . The method according to claim  149 , where the opioid is selected from the group consisting of morphine, methadone, etorphine, levorphanol, fentanyl, sufentanil, [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO), pentazocine, ethylketocyclazocine, bremazocine, spiradoline, [D-Ser2,Leu5]enkephalin-Thr6 (DSLET), Met-enkephalin, Leu-enkephalin, (3-endorphin, dynorphin A, dynorphin B, or a-neoendorphin.  
     
     
         31 . The method according to claim  149 , where the physiological effect is measured by changes in the levels of neuroendocrine hormones.  
     
     
         32 . The method according to claim  152 , where the hormone is selected from the group consisting of prolactin, growth hormone, gonadotropin-releasing hormone, adrenocorticotropin, corticotropin-releasing factor, luteinizing hormone, follicle stimulating hormone, testosterone or cortisol.  
     
     
         33 . A method of screening compositions for opioid binding activity comprising the steps of a) obtaining a control polypeptide that is not an MOR-1 splice variant polypeptide; b) obtaining a test polypeptide that is an MOR-1 splice variant polypeptide as in any one of claims  122 - 133 ; c) contacting a composition with the control polypeptide and the test polypeptide; d) contacting the test polypeptide with an amount of an opioid sufficient to measurably bind the test polypeptide; e) measuring the binding of the composition and the opioid; and f) comparing test polypeptide binding of the composition to that of the opioid, where determination of binding of the composition is expressed relative to that of the opioid.  
     
     
         34 . The method according to claim  154  where the composition is selected from the group consisting of synthetic combinatorial libraries of small molecule ligands, eukaryotic whole cell lysates or extracts, or media conditioned by cultured eukaryotic cells.  
     
     
         35 . A method for regulating morphine analgesia in a subject comprising altering the amount of MOR-1 polypeptide fragment activity by a) administering antigen binding fragments to a subject in an amount and a duration sufficient to regulate morphine analgesia; or b) administering agonists to a subject in an amount and a duration sufficient to regulate morphine analgesia; or c) administering antagonists to a subject in an amount and a duration sufficient to regulate morphine analgesia; or d) administering small molecule ligands to a subject in an amount and a duration sufficient to regulate morphine analgesia; and wherein the antigen binding fragment, agonist, antagonist or small molecule ligand is directed to an MOR-1 splice variant as in any one of claims  122 - 133 .