Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof

Abstract

The present invention relates to the field of GPCR structure biology and signaling. In particular, the present invention relates to protein binding domains directed against or capable of specifically binding to a functional conformational state of a G-protein-coupled receptor (GPCR). More specifically, the present invention provides protein binding domains that are capable of increasing the stability of a functional conformational state of a GPCR, in particular, increasing the stability of a GPCR in its active conformational state. The protein binding domains of the present invention can be used as a tool for the structural and functional characterization of G-protein-coupled receptors bound to various natural and synthetic ligands, as well as for screening and drug discovery efforts targeting GPCRs. Moreover, the invention also encompasses the diagnostic, prognostic and therapeutic usefulness of these protein binding domains for GPCR-related diseases.

1 .- 42 . (canceled) 
     
     
         43 . A cellular composition comprising a complex, the complex comprising:
 (i) a protein binding domain which specifically binds to an intracellular conformational epitope of a GPCR; and   (ii) (ii) the GPCR in a functional conformational state.   
     
     
         44 . The cellular composition of  claim 43 , wherein the complex further comprises a receptor ligand. 
     
     
         45 . The cellular composition of  claim 44 , wherein the receptor ligand is a full agonist, or a partial agonist, or an inverse agonist or an antagonist. 
     
     
         46 . The cellular composition of  claim 44 , in which said receptor ligand is a full agonist or a partial agonist. 
     
     
         47 . The cellular composition of  claim 44  wherein the receptor ligand is selected from the group consisting of a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate, and an antibody, or any suitable fragment thereof. 
     
     
         48 . The cellular composition of  claim 43 , wherein the protein binding domain specifically binds to a conformational epitope that is comprised in a binding site for a downstream signaling protein of the GPCR. 
     
     
         49 . The cellular composition of  claim 43 , wherein the protein binding domain specifically binds to a conformational epitope that is comprised in, located at or overlaps with the G protein binding site of the GPCR. 
     
     
         50 . The cellular composition of  claim 43 , wherein the protein binding domain protein occupies the G protein binding site of a functional conformational state of the GPCR. 
     
     
         51 . The cellular composition of  claim 43 , wherein the protein binding domain occupies the G protein binding site of an active conformational state of the GPCR. 
     
     
         52 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of the GPCR for an agonist. 
     
     
         53 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of the GPCR for an agonist at least twofold upon binding to the GPCR. 
     
     
         54 . The cellular composition of  claim 43 , wherein the protein binding domain (a) is derived from an immunoglobulin, (b) comprises an amino acid sequence comprising 4 framework regions and 3 complementary determining regions or any suitable fragment thereof, (c) is derived from a camelid antibody, and/or (d) comprises a nanobody sequence or any suitable fragment thereof. 
     
     
         55 . The cellular composition of  claim 43 , wherein the cellular composition is an organism, a tissue, a cell, a cell line, a membrane composition, or a liposomal composition. 
     
     
         56 . The cellular composition of  claim 55 , wherein the cellular composition is a cell or a cell line. 
     
     
         57 . The cellular composition of  claim 55 , wherein the cellular composition is a membrane composition or a liposomal composition. 
     
     
         58 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of the GPCR for an agonist at least fivefold. 
     
     
         59 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of the GPCR for an agonist at least tenfold.