Methods for screening for binding partners of g-protein coupled receptors

Abstract

A method of producing a conformational specific binding partner of a GPCR, the method comprising: a) providing a mutant GPCR of a parent GPCR, wherein the mutant GPCR has increased stability in a particular conformation relative to the parent GPCR; b) providing a test compound; c) determining whether the test compound binds to the mutant GPCR when residing in a particular conformation; and d) isolating a test compound that binds to the mutant GPCR when residing in the particular formation. Methods of producing GPCRs with increased stability relative to a parent GPCR are also disclosed.

1 - 108 . (canceled) 
     
     
         109 . A mutant human G-protein coupled receptor (GPCR) with increased conformational stability in a particular conformation compared to its parent human GPCR in the same particular conformation. 
     
     
         110 . The mutant human GPCR of  claim 109  which has increased conformational stability in an agonist conformation compared to its parent human GPCR in an agonist conformation. 
     
     
         111 . The mutant human GPCR of  claim 109  which has increased conformational stability in an antagonist conformation compared to its parent human GPCR in an antagonist conformation. 
     
     
         112 . The mutant human GPCR of  claim 109 , wherein the mutant GPCR has increased binding retention to a ligand of a particular class under denaturing conditions. 
     
     
         113 . The mutant human GPCR of  claim 112 , wherein the denaturing conditions are selected from the group consisting of heat, a detergent, a chaotropic agent and an extreme of pH. 
     
     
         114 . The mutant human GPCR of  claim 109 , wherein the mutant differs from its parent human GPCR by one or more point mutations. 
     
     
         115 . The mutant GPCR of  claim 109 , which contains, compared to its parent GPCR, from 1 to 10 replaced amino acids. 
     
     
         116 . The mutant human GPCR of  claim 109 , wherein the mutant human GPCR has increased conformational thermostability relative to its parent human GPCR. 
     
     
         117 . The mutant human GPCR of  claim 109 , wherein the mutant human GPCR is selected from the group consisting of: a mutant adenosine receptor, a mutant β-adrenergic receptor, a mutant neurotensin receptor, a mutant muscarinic acid receptor, a mutant 5-hydroxytryptamine receptor, a mutant adrenoceptor, a mutant anaphylatoxin receptor, a mutant angiotensin receptor, a mutant apelin receptor, a mutant bombesin receptor, a mutant bradykinin receptor, a mutant cannabinoid receptor, a mutant chemokine receptor, a mutant cholecystokinin receptor, a mutant dopamine receptor, a mutant endothelin receptor, a mutant free fatty acid receptor, a mutant bile acid receptor, a mutant galanin receptor, a mutant motilin receptor, a mutant ghrelin receptor, a mutant glycoprotein hormone receptor, a mutant GnRH receptor, a mutant histamine receptor, a mutant KiSS1-derived peptide receptor, a mutant leukotriene and lipoxin receptor, a mutant lysophospholipid receptor, a mutant melanin-concentrating hormone receptor, a mutant melanocortin receptor, a mutant melatonin receptor, a mutant neuromedin U receptor, a mutant neuropeptide receptor, a mutant N-formylpeptide family receptor, a mutant nicotinic acid receptor, a mutant opiod receptor, a mutant opsin-like receptor, a mutant orexin receptor, a mutant P2Y receptor, a mutant peptide P518 receptor, a mutant platelet-activating factor receptor, a mutant prokineticin receptor, a mutant prolactin-releasing peptide receptor, a mutant prostanoid receptor, a mutant protease-activated receptor, a mutant relaxin receptor, a mutant somatostatin receptor, a mutant SPC/LPC receptor, a mutant tachykinin receptor, a mutant trace amino receptor, a mutant thryotropin-releasing hormone receptor, a mutant urotensin receptor, a mutant vasopressin/oxytocin receptor, a mutant orphan GPCR, a mutant calcitonin receptor, a mutant corticotropin releasing factor receptor, a mutant glucagon receptor, a mutant parathyroid receptor, a mutant VIP/PACAP receptor, a mutant LNB7TM receptor, a mutant GABA receptor, a mutant metabotropic glutamate receptor, and a mutant calcium sensor receptor. 
     
     
         118 . The mutant human GPCR of  claim 109 , wherein the mutant human GPCR is a muscarinic receptor. 
     
     
         119 . A mutant human G-protein coupled receptor (GPCR) with increased conformational stability compared to its parent GPCR, which mutant GPCR has an extended lifetime of a particular conformation relative to the same conformation of its parent GPCR under denaturing conditions, the extended lifetime being manifest by retention of ligand binding ability. 
     
     
         120 . A mutant human GPCR of  claim 119 , wherein the particular conformation is selected from an agonist conformation and an antagonist conformation, and
 wherein the mutant human GPCR has increased binding retention to a ligand of a particular class under denaturing conditions, wherein:   (i) a mutant human GPCR with increased conformational stability in an agonist conformation has increased binding retention to an agonist ligand; and   (ii) a mutant human GPCR with increased conformational stability in an antagonist conformation has increased binding retention to an antagonist ligand.   
     
     
         121 . The mutant GPCR of  claim 119 , wherein the mutant human GPCR differs from its parent human GPCR by one or more point mutations. 
     
     
         122 . The mutant GPCR of  claim 119  which contains, compared to its parent GPCR, from 1 to 10 replaced amino acids. 
     
     
         123 . The mutant GPCR of  claim 119 , wherein the denaturing conditions are selected from the group consisting of heat, a detergent, a chaotropic agent, and an extreme of pH. 
     
     
         124 . The mutant human GPCR of  claim 119 , wherein the mutant human GPCR has increased conformational thermostability relative to its parent human GPCR. 
     
     
         125 . The mutant human GPCR of  claim 119 , wherein the mutant human GPCR is selected from the group consisting of: a mutant adenosine receptor, a mutant β-adrenergic receptor, a mutant neurotensin receptor, a mutant muscarinic acid receptor, a mutant 5-ydroxytryptamine receptor, a mutant adrenoceptor, a mutant anaphylatoxin receptor, a mutant angiotensin receptor, a mutant apelin receptor, a mutant bombesin receptor, a mutant bradykinin receptor, a mutant cannabinoid receptor, a mutant chemokine receptor, a mutant cholecystokinin receptor, a mutant dopamine receptor, a mutant endothelin receptor, a mutant free fatty acid receptor, a mutant bile acid receptor, a mutant galanin receptor, a mutant motilin receptor, a mutant ghrelin receptor, a mutant glycoprotein hormone receptor, a mutant GnRH receptor, a mutant histamine receptor, a mutant KiSS1-derived peptide receptor, a mutant leukotriene and lipoxin receptor, a mutant lysophospholipid receptor, a mutant melanin-concentrating hormone receptor, a mutant melanocortin receptor, a mutant melatonin receptor, a mutant neuromedin U receptor, a mutant neuropeptide receptor, a mutant N-formylpeptide family receptor, a mutant nicotinic acid receptor, a mutant opiod receptor, a mutant opsin-like receptor, a mutant orexin receptor, a mutant P2Y receptor, a mutant peptide P518 receptor, a mutant platelet-activating factor receptor, a mutant prokineticin receptor, a mutant prolactin-releasing peptide receptor, a mutant prostanoid receptor, a mutant protease-activated receptor, a mutant relaxin receptor, a mutant somatostatin receptor, a mutant SPC/LPC receptor, a mutant tachykinin receptor, a mutant trace amino receptor, a mutant thryotropin-releasing hormone receptor, a mutant urotensin receptor, a mutant vasopressin/oxytocin receptor, a mutant orphan GPCR, a mutant calcitonin receptor, a mutant corticotropin releasing factor receptor, a mutant glucagon receptor, a mutant parathyroid receptor, a mutant VIP/PACAP receptor, a mutant LNB7TM receptor, a mutant GABA receptor, a mutant metabotropic glutamate receptor, and a mutant calcium sensor receptor. 
     
     
         126 . The mutant human GPCR of  claim 119 , wherein the mutant human GPCR is a muscarinic receptor. 
     
     
         127 . A mutant G-protein coupled receptor (GPCR) with increased conformational stability in a particular conformation compared to its parent GPCR in the same particular conformation, and wherein the mutant is not a mutant rat neurotensin receptor, which, when compared to its parent rat neurotensin receptor, contains an F358A mutation according to the numbering of the rat neurotensin receptor as set forth in SEQ ID NO: 9. 
     
     
         128 . The mutant GPCR of  claim 127 , wherein the which particular conformation is selected from an agonist conformation and an antagonist conformation. 
     
     
         129 . A mutant G-protein coupled receptor (GPCR) with increased conformational stability compared to its parent GPCR which mutant PCR has an extended lifetime of a particular conformation relative to the same conformation of its parent GPCR under denaturing conditions, the extended lifetime being manifest by retention of ligand binding ability, and wherein the mutant is not a mutant rat neurotensin receptor, which, when compared to its parent rat neurotensin receptor, contains an F358A mutation according to the numbering of the rat neurotensin receptor as set forth in SEQ ID NO: 9. 
     
     
         130 . The mutant GPCR of  claim 129 , wherein the particular conformation is selected from an agonist conformation and an antagonist conformation,
 wherein the mutant GPCR has increased binding retention to a ligand of a particular class under denaturing conditions, wherein:   (i) a mutant GPCR with increased conformational stability in an agonist conformation has increased binding retention to an agonist ligand; and   (ii) a mutant GPCR with increased conformational stability in an antagonist conformation has increased binding retention to an antagonist ligand.