Mutant g-protein coupled receptors and methods for selecting them

Abstract

The invention relates to mutant G-protein coupled receptors with increased conformational stability, and methods of use thereof. In some aspects, polynucleotides encoding the mutant G-protein coupled receptors are provided. In some aspects, host cells comprising the polynucleotides are provided. In some aspects, the invention relates to crystallized forms of the mutant G-protein coupled receptors, and methods of preparing the same.

1 . A mutant 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. 
     
     
         2 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR has an extended lifetime under conditions selected from any one or more of heat, a detergent, a chaotropic agent and an extreme of pH. 
     
     
         3 . A mutant GPCR according to  claim 2 , wherein the mutant GPCR has increased conformational thermostability, optionally which is at least 1° C. more stable than its parent. 
     
     
         4 . A mutant GPCR according to  claim 1 , with increased conformational stability of an agonist or antagonist conformation. 
     
     
         5 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR is a Class 1 GPCR. 
     
     
         6 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR is a Class 2 GPCR. 
     
     
         7 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR is a Class 3 GPCR. 
     
     
         8 . A polynucleotide encoding a mutant GPCR according to  claim 1 . 
     
     
         9 . A host cell comprising a polynucleotide according to  claim 8 . 
     
     
         10 . A mutant GPCR according to  claim 1 , which is in a solubilized form. 
     
     
         11 . A mutant GPCR according to  claim 1 , which is substantially free of other proteins. 
     
     
         12 . A mutant GPCR according to  claim 1 , which is immobilized to a solid support. 
     
     
         13 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR is not a human cannabinoid receptor. 
     
     
         14 . A polynucleotide according to  claim 8 , wherein the mutant GPCR is not a human cannabinoid receptor. 
     
     
         15 . A host cell according to  claim 9 , wherein the mutant GPCR is not a human cannabinoid receptor. 
     
     
         16 . A crystallized form of a mutant GPCR according to  claim 1 . 
     
     
         17 . A method comprising crystallizing a mutant GPCR of  claim 1 . 
     
     
         18 . A method comprising
 obtaining a mutant GPCR of  claim 1 , and   screening for a ligand that binds to the mutant GPCR.   
     
     
         19 . The method of  claim 18 , wherein the mutant GPCR is coupled to a solid surface of a biosensor chip. 
     
     
         20 . A method comprising
 obtaining a mutant GPCR of  claim 1 , and   developing an assay involving binding of a ligand to the mutant GPCR.