Receptor tyrosine kinase assays

Abstract

Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation and the modulation of activation by a candidate compound are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of β-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of β-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a β-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

What is claimed is: 
     
         1 . A method for determining an effect of a candidate compound on phosphorylation of a receptor tyrosine kinase (“RTK”) said method comprising:
 (a) providing a cell comprising (i) a first expression construct expressing a fusion of an RTK fused to a first enzyme fragment and (ii) a second expression construct expressing a fusion of a phosphotyrosine binding peptide fused to a second enzyme fragment, 
 (b) wherein said first enzyme fragment and said second enzyme fragment are fragments of β-galactosidase that have low affinity for each other but when brought together by the binding of said RTK to said phosphotyrosine binding peptide form an active β-galactosidase, with the proviso that when said RTK does not autophosphorylate, in the absence of an endogenous active cytosolic tyrosine kinase, a third expression construct is included expressing a cytosolic tyrosine kinase to phosphorylate said RTK; 
 (c) contacting said cell with said candidate compound; 
 (d) incubating said cell for sufficient time for any phosphorylation to occur; 
 (e) adding a β-galactosidase substrate to said cell, wherein said substrate forms a detectable product; and 
 (f) detecting a product formed as indicative of the effect of the candidate compound on the phosphorylation of said RTK. 
 
     
     
         2 . A method according to  claim 1 , wherein said first enzyme fragment is the small fragment of β-galactosidase having fewer than 50 amino acids. 
     
     
         3 . A method according to  claim 2 , wherein said small fragment is mutated with respect to native β-galactosidase. 
     
     
         4 . A method according to  claim 1 , wherein said cell is a mammalian cell. 
     
     
         5 . A method according to  claim 1 , including the additional step of lysing said cell before said detecting. 
     
     
         6 . A method according to  claim 1 , wherein said product is chemiluminescent. 
     
     
         7 . A method according to  claim 1 , wherein said candidate compound is tested as an antagonist, wherein a ligand for said RTK is added prior to addition of said candidate compound. 
     
     
         8 . A method for determining presence of active ligand for receptor tyrosine kinase (“RTK”) in a sample, comprising:
 (a) providing a cell comprising (i) a first expression construct expressing a fusion of an RTK fused at its C-terminus to a first enzyme fragment and (ii) a second expression construct expressing a fusion of a phosphotyrosine binding peptide fused to a second enzyme fragment; 
 (b) wherein said first enzyme fragment and said second enzyme fragment are fragments of β-galactosidase that have low affinity for each other but when brought together by the binding of said RTK to said phosphotyrosine binding peptide form an active β-galactosidase, with the proviso that when said RTK does not autophosphorylate, in the absence of an endogenous active cytosolic tyrosine kinase, a third expression construct is included expressing a cytosolic tyrosine kinase to phosphorylate said RTK; 
 (c) contacting said cell with said sample; 
 (d) incubating said cell for sufficient time for any phosphorylation to occur; 
 (e) adding a β-galactosidase substrate to said cell, wherein said substrate forms a detectable product; and 
 (f) detecting a product formed as indicative of the presence of an active ligand for said RTK. 
 
     
     
         9 . A method according to  claim 8 , wherein said first enzyme fragment is the small fragment of β-galactosidase having fewer than 50 amino acids. 
     
     
         10 . A method according to  claim 9 , wherein said small fragment is mutated with respect to native β-galactosidase. 
     
     
         11 . A method according to  claim 8 , wherein said cell is a mammalian cell. 
     
     
         12 . A method according to  claim 8 , including the additional step of lysing said cell before said detecting. 
     
     
         13 . A method according to  claim 8 , wherein said product is chemiluminescent.