Assay for monitoring activity of frizzled receptors

Abstract

The present invention relates to cell free assays for measuring receptor activity, especially for measuring a constitutive or a non-constitutive activity of frizzled receptors and uses thereof. The present invention further concerns a method for measuring a constitutive or non-constitutive activity of a frizzled receptor and a method for obtaining an active frizzled receptor ligand.

1 - 6 . (canceled) 
     
     
         7 . A method for measuring a constitutive activity of a frizzled receptor comprising the steps of:
 a) providing at least one membrane protein with at least one cell free frizzled receptor;   b) adding at least one frizzled receptor ligand and at least one G protein; and   c) incubating the cell free frizzled receptor, the frizzled receptor ligand and the G protein in a system for measuring G protein activity,   
       wherein the detection of guanosine triphosphate (GTP) binding to the G protein indicates the activity of the G protein. 
     
     
         8 . The method of  claim 7 , wherein a concentration of the G protein is approximately 0.4 μg/ml or less. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 7 , wherein the frizzled receptor ligand is a Wnt protein. 
     
     
         11 . The method of  claim 7 , wherein step a) and/or step b) are/is preceded by at least one protein evolution technique. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The method of  claim 7 , wherein the frizzled receptor ligands are drug candidates. 
     
     
         15 . The method of  claim 14 , wherein the drug candidates are for cancer. 
     
     
         16 . The method of  claim 14 , wherein the drug candidates are for degenerative disorders selected from the group consisting of tetra-amelia, bone density defects, vascular and retinal defects in the eye, and tooth agenesis. 
     
     
         17 . A method for measuring a non-constitutive activity of a frizzled receptor comprising the steps of:
 a) providing at least one membrane protein with at least one cell-free frizzled receptor;   b) adding at least one frizzled receptor ligand and at least one G protein; and   c) incubating the cell-free frizzled receptor, the frizzled receptor ligand and the G protein in a system for measuring G protein activity,   
       wherein the detection of guanosine triphosphate (GTP) binding to the G protein indicates the activity of the G protein. 
     
     
         18 . The method of  claim 17 , wherein a concentration of the G protein is approximately 0.3 μg/ml or higher. 
     
     
         19 . The method of  claim 17 , wherein the frizzled receptor ligand is a Wnt protein. 
     
     
         20 . The method of  claim 17 , wherein step a) and/or step b) are/is preceded by at least one protein evolution technique. 
     
     
         21 . The method of  claim 17 , wherein the frizzled receptor ligands are drug candidates. 
     
     
         22 . The method of  claim 21 , wherein the drug candidates are for cancer. 
     
     
         23 . The method of  claim 21 , wherein the drug candidates are for degenerative disorders selected from the group consisting of tetra-amelia, bone density defects, vascular and retinal defects in the eye, and tooth agenesis.