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

1. 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, which particular conformation is selected from an agonist conformation and an antagonist conformation, produced by a method comprising:

(a) providing one or more mutants of a parent human GPCR;

(b) contacting the one or more mutants and the parent human GPCR with a ligand of a particular class, wherein:

(i) an agonist ligand is used to select for one or more mutants with increased conformational stability in the agonist conformation; and

(ii) an antagonist ligand is used to select for one or more mutants with increased conformational stability in the antagonist conformation;

(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the selected ligand compared to the stability of the parent human GPCR with respect to binding the selected ligand, and

(d) selecting the one or more mutants that exhibit increased stability compared to the parent human GPCR with respect to binding the selected ligand.

2. A mutant human G-protein coupled receptor (GPCR) with increased conformational stability in an agonist conformation compared to its parent human GPCR in an agonist conformation, produced by a method comprising:

(a) providing one or more mutants of a parent human GPCR;

(b) contacting the one or more mutants and the parent human GPCR with an agonist ligand;

(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the agonist ligand compared to the parent human GPCR with respect to binding the agonist ligand, and

(d) selecting those mutants that have an increased stability compared to the parent human GPCR with respect to binding of the agonist ligand.

3. The mutant human GPCR of claim 2 , wherein the one or more mutants each differs from its parent human GPCR by one or more point mutations.

4. The mutant human GPCR of claim 2 , wherein the mutant human GPCR has increased conformational thermostability relative to its parent human GPCR.

5. The mutant human GPCR of claim 2 , 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.

6. The mutant human GPCR of claim 2 , wherein the mutant human GPCR is a muscarinic receptor.

7. A mutant human G-protein coupled receptor (GPCR) with increased conformational stability in an antagonist conformation compared to its parent human GPCR in an antagonist conformation, produced by a method comprising:

(a) providing one or more mutants of a parent human GPCR;

(b) contacting the one or more mutants and the parent human GPCR with an antagonist ligand;

(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the antagonist ligand compared to the parent human GPCR with respect to binding the antagonist ligand, and

(d) selecting those mutants that have an increased stability compared to the parent human GPCR with respect to binding of the antagonist ligand.

8. The mutant human GPCR of claim 7 , wherein the one or more mutants each differs from its parent human GPCR by one or more point mutations.

9. The mutant human GPCR of claim 7 , wherein the mutant human GPCR has increased conformational thermostability relative to its parent human GPCR.

10. The mutant human GPCR of claim 7 , 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.

11. The mutant human GPCR of claim 7 , wherein the mutant human GPCR is a muscarinic receptor.

12. 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, which particular conformation is selected from an agonist conformation and an antagonist conformation,

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; and

wherein the mutant human GPCR differs from its parent human GPCR by one or more point mutations in a structural motif of the mutant human GPCR selected from the group consisting of a helical interface, a helix kink, a helix opposite a helix kink, a helix surface pointing into the lipid bilayer, a helix surface pointing into the lipid bilayer at the hydrophobic-hydrophilic boundary layer, a loop region and a protein binding pocket.

13. The mutant human GPCR of claim 12 , wherein the mutant human GPCR has increased conformational thermostability relative to its parent human GPCR.

14. The mutant human GPCR of claim 12 , 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.

15. The mutant human GPCR of claim 12 , wherein the mutant human GPCR is a muscarinic receptor.

16. 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, which particular conformation is selected from an agonist conformation and an antagonist conformation, produced by a method comprising:

(a) providing one or more mutants of a parent GPCR;

(b) contacting the one or more mutants and the parent GPCR with a ligand of a particular class, wherein:

(i) an agonist ligand is used to select for one or more mutants with increased conformational stability in the agonist conformation; and

(ii) an antagonist ligand is used to select for one or more mutants with increased conformational stability in the antagonist conformation;

(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the selected ligand compared to the stability of the parent GPCR with respect to binding the selected ligand, and

(d) selecting the one or more mutants that exhibit increased stability compared to the parent GPCR with respect to binding the selected ligand,

wherein the selected one or more mutants are not mutant rat neurotensin receptors, which, when compared to their parent rat neurotensin receptor, contain an F358A mutation according to the numbering of the rat neurotensin receptor as set forth in SEQ ID NO: 9.

17. A mutant G-protein coupled receptor (GPCR) with increased conformational stability in an agonist conformation compared to its parent GPCR in an agonist conformation, produced by a method comprising:

(a) providing one or more mutants of a parent GPCR;

(b) contacting the one or more mutants and the parent GPCR with an agonist ligand;

(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the agonist ligand compared to the parent GPCR with respect to binding the agonist ligand, and

(d) selecting those mutants that have an increased stability compared to the parent GPCR with respect to binding of the agonist ligand,

wherein the selected mutants are not mutant rat neurotensin receptors, which, when compared to their parent rat neurotensin receptor, contain an F358A mutation according to the numbering of the rat neurotensin receptor as set forth in SEQ ID NO: 9.

18. A mutant G-protein coupled receptor (GPCR) with increased conformational stability in an antagonist conformation compared to its parent GPCR in an antagonist conformation, produced by a method comprising:

(a) providing one or more mutants of a parent GPCR;

(b) contacting the one or more mutants and the parent GPCR with an antagonist ligand;

(c) determining whether the one or more mutant GPCRs has increased stability with respect to binding the antagonist ligand compared to the parent GPCR with respect to binding the antagonist ligand, and

(d) selecting those mutants that have an increased stability compared to the parent GPCR with respect to binding of the antagonist ligand,

wherein the selected mutants are not mutant rat neurotensin receptors, which, when compared to their parent rat neurotensin receptor, contain an F358A mutation according to the numbering of the rat neurotensin receptor as set forth in SEQ ID NO: 9.

19. 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, which 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; and

wherein the mutant GPCR differs from its parent GPCR by one or more point mutations in a structural motif of the mutant GPCR selected from the group consisting of a helical interface, a helix kink, a helix opposite a helix kink, a helix surface pointing into the lipid bilayer, a helix surface pointing into the lipid bilayer at the hydrophobic-hydrophilic boundary layer, a loop region and a protein binding pocket, and

wherein the mutant GPCR 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.