Reversed mammalian protein-protein interaction trap

Abstract

The present invention relates to a recombinant receptor, comprising a ligand-binding domain and a signaling domain that comprises a heterologous bait polypeptide, which receptor is inactivated by binding of a prey polypeptide to the heterologous bait peptide, either in presence or absence of a ligand binding to the ligand-binding domain. The receptor is activated by addition of a compound that disrupts the bait-prey interaction. The present invention also relates to a method of screening compounds that disrupt compound-compound-binding using the recombinant receptor.

What is claimed is:  
     
         1 . A recombinant receptor comprising: 
 a ligand-binding domain and    a domain that comprises a heterologous bait polypeptide,    wherein the activation of said recombinant receptor is inhibited by binding of a prey polypeptide to said heterologous bait peptide.    
     
     
         2 . The recombinant receptor of  claim 1 , wherein said recombinant receptor is a transmembrane receptor.  
     
     
         3 . The recombinant receptor of  claim 1  or  claim 2 , wherein said recombinant receptor is activated by the addition of a compound that disrupts the bait-prey interaction.  
     
     
         4 . The recombinant receptor  claim 1 ,  claim 2 , or  claim 3  wherein said recombinant receptor is a homomultimerizing receptor.  
     
     
         5 . The recombinant receptor of claims  1 ,  claim 2 , or  claim 3  wherein said recombinant receptor is a heteromultimerizing receptor.  
     
     
         6 . The recombinant receptor of  claim 1 ,  claim 2 ,  claim 3 ,  claim 4 , or  claim 5  wherein the binding of said prey polypeptide depends upon the modification state of said heterologous bait peptide.  
     
     
         7 . The recombinant receptor of  claim 6  wherein the modification state is presence or absence of phosphorylation, acetylation, acylation, methylation, ubiquitinilation or glycosylation.  
     
     
         8 . The recombinant receptor of  claim 6  or  claim 7  wherein the change of the modification state is dependent upon binding of a ligand to the ligand-binding domain.  
     
     
         9 . A prey polypeptide comprising: 
 a polypeptide that interacts with a bait polypeptide and    a polypeptide comprising an inhibitor of activation of a receptor and/or a recruitment site for an inhibitor of activation of a receptor.    
     
     
         10 . The prey polypeptide of  claim 9 , comprising: 
 a polypeptide that interacts with the heterologous bait polypeptide of the recombinant receptor of  claim 1 ,  claim 2 ,  claim 3 ,  claim 4 ,  claim 5 ,  claim 6 ,  claim 7 , or  claim 8  and a polypeptide comprising an inhibitor of a receptor.    
     
     
         11 . A vector encoding the recombinant receptor of  claim 1 ,  claim 2 ,  claim 3 ,  claim 4 ,  claim 5 ,  claim 6 ,  claim 7 , or  claim 8 .  
     
     
         12 . A vector encoding the prey polypeptide of  claim 9  or  claim 10 .  
     
     
         13 . A eukaryotic cell comprising the recombinant receptor of  claim 1 ,  claim 2 ,  claim 3 ,  claim 4 ,  claim 5 ,  claim 6 ,  claim 7 , or  claim 8 .  
     
     
         14 . A eukaryotic cell comprising the prey polypeptide of  claim 9  or  claim 10 .  
     
     
         15 . The eukaryotic cell of  claim 13  or  claim 14 , where said cell is selected from the group consisting of a mammalian cell, a fungal cell, and a plant cell.  
     
     
         16 . A kit, comprising a cloning vector allowing the construction of the vector of  claim 11  or  claim 12 .  
     
     
         17 . A method of screening compounds that disrupt compound-compound binding, said method comprising: 
 screening compounds with the recombinant receptor of  claim 1 ,  claim 2 ,  claim 3 ,  claim 4 ,  claim 5 ,  claim 6 ,  claim 7 , or  claim 8  and/or a prey polypeptide comprising a polypeptide that interacts with a bait polypeptide and a polypeptide comprising an inhibitor of activation of a receptor and/or a recruitment site for an inhibitor of activation of a receptor.    
     
     
         18 . The method according to  claim 17 , wherein said compound-compound binding is modification state dependent.  
     
     
         19 . The method according to  claim 18 , wherein said modification is phosphorylation, acetylation, acylation, methylation, ubiquitinilation or glycosylation.  
     
     
         20 . The method according to  claim 17 ,  claim 18 , or  claim 19 , wherein said binding is mediated by three or more partners.  
     
     
         21 . The method according to  claim 20 , wherein at least one of the partners is not or not completely of proteinaceous nature.