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 . Cellular composition comprising a protein binding domain that specifically binds to an intracellular conformational epitope of a GPCR. 
     
     
         44 . The cellular composition of  claim 43 , wherein the protein binding domain stabilizes and/or induces a functional conformational state of the GPCR upon binding of the protein binding domain to the GPCR. 
     
     
         45 . The cellular composition of  claim 43 , wherein the protein binding domain specifically binds a conformational epitope of the GPCR that is comprised in a binding site for a downstream signaling protein. 
     
     
         46 . The cellular composition of  claim 43 , wherein the protein binding domain specifically binds a conformational epitope that is comprised in, located at or overlaps with the G protein binding site of the GPCR. 
     
     
         47 . 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 when bound to the GPCR. 
     
     
         48 . 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 when bound to the GPCR. 
     
     
         49 . The cellular composition of  claim 43 , wherein the protein binding domain protein preferentially binds agonist-bound receptor versus inverse agonist-bound receptor. 
     
     
         50 . The cellular composition of  claim 43 , wherein the protein binding domain is capable of specifically binding to an agonist-bound GPCR. 
     
     
         51 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of a receptor for an agonist. 
     
     
         52 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of a GPCR for an agonist at least twofold upon binding to the GPCR. 
     
     
         53 . 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. 
     
     
         54 . 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 
     
     
         55 . The cellular composition of  claim 54 , wherein the cellular composition is a cell or a cell line. 
     
     
         56 . The cellular composition of  claim 54 , wherein the cellular composition is a membrane composition or a liposomal composition. 
     
     
         57 . A method of capturing a GPCR in a functional conformational state, the method comprising:
 contacting the GPCR with the cellular composition of  claim 43 ; and   forming a complex of the GPCR and the protein binding domain.   
     
     
         58 . A method of capturing a GPCR in an active conformational state, the method comprising:
 contacting the GPCR with the cellular composition of  claim 43 ; and   forming a complex of the GPCR and the protein binding domain.   
     
     
         59 . A method of capturing a GPCR in a functional conformational state with a receptor ligand, the method comprising:
 contacting the GPCR with the cellular composition of  claim 43  and the receptor ligand; and   forming a complex of the GPCR, the receptor ligand and the protein binding domain.   
     
     
         60 . A method of capturing a GPCR in an active conformational state with a receptor ligand, the method comprising:
 contacting the GPCR with the cellular composition of  claim 43  and the receptor ligand; and   forming a complex of the GPCR, the receptor ligand and the protein binding domain.   
     
     
         61 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of the GPCR for an agonist at least fivefold. 
     
     
         62 . The cellular composition of  claim 43 , wherein the protein binding domain enhances the affinity of the GPCR for an agonist at least tenfold.