Kinase receptor activation assay

Abstract

An assay for measuring activation (i.e., autophosphorylation) of a tyrosine kinase receptor of interest is disclosed. (a) A first solid phase is coated with a substantially homogeneous population of cells so that the cells adhere to the first solid phase. The cells have either an endogenous tyrosine kinase receptor or have been transformed with DNA encoding a receptor or “receptor construct” and the DNA has been expressed so that the receptor or receptor construct is presented in the cell membranes of the cells. (b) A ligand is then added to the solid phase having the adhering cells, such that the tyrosine kinase receptor is exposed to the ligand. (c) Following exposure to the ligand, the adherent cells are solubilized, thereby releasing cell lysate. (d) A second solid phase is coated with a capture agent which binds specifically to the tyrosine kinase receptor, or, in the case of a receptor construct, to the flag polypeptide. (e) The cell lysate obtained in step (c) is added to the wells containing the adhering capture agent so as to capture the receptor or receptor construct to the wells. (f) A washing step is then carried out, so as to remove unbound cell lysate, leaving the captured receptor or receptor construct. (g) The captured receptor or receptor construct is exposed to a labelled anti-phosphotyrosine antibody which identifies phosphorylated residues in the tyrosine kinase receptor. (h) Binding of the anti-phosphotyrosine antibody to the captured receptor or receptor construct is measured.

1 . A method for measuring autophosphorylation of a tyrosine kinase receptor comprising the steps of: 
 (a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein, positioned in their membranes, the cells have a receptor construct comprising a flag polypeptide and the tyrosine kinase receptor;    (b) exposing the adhering cells to an analyte;    (c) solubilizing the adhering cells, thereby releasing cell lysate therefrom;    (d) coating a second solid phase with a capture agent which binds specifically to the flag polypeptide so that the capture agent adheres to the second solid phase;    (e) exposing the adhering capture agent to the cell lysate obtained in step (c) so that the receptor construct adheres to the second solid phase;    (f) washing the second solid phase so as to remove unbound cell lysate;    (g) exposing the adhering receptor construct to an anti-phosphotyrosine antibody which identifies phosphorylated tyrosine residues in the tyrosine kinase receptor; and    (h) measuring binding of the anti-phosphotyrosine antibody to the adhering receptor construct.    
     
     
         2 . The method of  claim 1  wherein the cells are transformed with nucleic acid encoding the receptor construct prior to step (a).  
     
     
         3 . The method of  claim 1  wherein the cells comprise a mammalian cell line.  
     
     
         4 . The method of  claim 1  wherein the cells are adherent.  
     
     
         5 . The method of  claim 1  wherein the capture agent comprises a capture antibody.  
     
     
         6 . The method of  claim 1  wherein the first solid phase comprises a well of a first assay plate.  
     
     
         7 . The method of  claim 6  wherein the first assay plate is a microtiter plate.  
     
     
         8 . The method of  claim 6  wherein between about 1×10 4  to 3×10 5  cells are added to the well in step (a).  
     
     
         9 . The method of  claim 1  wherein the second solid phase comprises a well of a second assay plate.  
     
     
         10 . The method of  claim 1  wherein the cell lysate is not concentrated or clarified prior to step (e).  
     
     
         11 . The method of  claim 6  wherein step (c) comprises adding a lysis buffer to the well of the first assay plate and gently agitating the first assay plate.  
     
     
         12 . The method of  claim 11  wherein the lysis buffer comprises a solubilizing detergent.  
     
     
         13 . The method of  claim 1  wherein the anti-phosphotyrosine antibody is labelled.  
     
     
         14 . The method of  claim 13  wherein the label comprises an enzyme which is exposed to a color reagent and the color change of the color reagent is determined in step (h).  
     
     
         15 . The method of  claim 1  wherein the flag polypeptide is fused to the amino terminus of the tyrosine kinase receptor.  
     
     
         16 . The method of  claim 15  wherein the tyrosine kinase receptor is a trk A receptor, trk B receptor or trk C receptor.  
     
     
         17 . The method of  claim 1  wherein the flag polypeptide is fused to the carboxyl terminus of the tyrosine kinase receptor.  
     
     
         18 . The method of  claim 17  wherein the tyrosine kinase receptor is the Rse receptor.  
     
     
         19 . The method of  claim 17  wherein the receptor construct comprises the extracellular domain of a receptor of interest and the intracellular domain of the Rse receptor.  
     
     
         20 . The method of  claim 19  wherein the receptor of interest is the MPL receptor.  
     
     
         21 . The method of  claim 20  wherein the receptor construct further comprises the transmembrane domain of the Rse receptor and the flag polypeptide comprises the gD polypeptide.  
     
     
         22 . The method of  claim 1  wherein the analyte comprises an agonist for the tyrosine kinase receptor.  
     
     
         23 . The method of  claim 1  wherein the analyte comprises an antagonist for the tyrosine kinase receptor.  
     
     
         24 . The method of  claim 23  wherein the antagonist competitively inhibits binding or activation of the tyrosine kinase receptor by an agonist thereto and step (b) is followed by a step wherein the adhering cells are exposed to the agonist.  
     
     
         25 . The method of  claim 1  wherein the analyte is a composition which comprises an antagonist and an agonist for the receptor and the assay measures the ability of the antagonist to bind to the agonist and thereby reduce activation of the tyrosine kinase receptor by the agonist.  
     
     
         26 . The method of  claim 1  wherein a block buffer is added to the second solid phase following step (d).  
     
     
         27 . A method for measuring autophosphorylation of a tyrosine kinase receptor comprising the steps of: 
 (a) coating a well of a first assay plate with a homogeneous population of adherent cells so that the cells adhere to the well, wherein the cells have a tyrosine kinase receptor positioned in the cell membranes thereof;    (b) exposing the adhering cells to an analyte;    (c) solubilizing the adhering cells thereby releasing cell lysate therefrom;    (d) coating a well of a second assay plate with a capture agent which binds specifically to the tyrosine kinase receptor so that the capture agent adheres to the well;    (e) exposing the cell lysate obtained in step (c) to the adhering capture agent so that the tyrosine kinase receptor adheres to the well;    (f) washing the well so as to remove unbound cell lysate;    (g) exposing the adhering tyrosine kinase receptor to an anti-phosphotyrosine antibody which binds selectively to phosphorylated tyrosine residues in the tyrosine kinase receptor;    (h) measuring binding of the anti-phosphotyrosine antibody to the adhering tyrosine kinase receptor.    
     
     
         28 . The method of  claim 27  wherein the tyrosine kinase receptor comprises the HER2 receptor.  
     
     
         29 . A polypeptide comprising a flag polypeptide fused to the carboxyl terminus of the intracellular domain of the Rse receptor.  
     
     
         30 . The polypeptide of  claim 29  further comprising the transmembrane domain of the Rse receptor.  
     
     
         31 . The polypeptide of  claim 30  further comprising the extracellular domain of a receptor protein tyrosine kinase, other than the Rse receptor.  
     
     
         32 . The polypeptide of  claim 29  wherein the flag polypeptide comprises the gD flag.  
     
     
         33 . A kit comprising a solid phase coated with a capture agent which binds specifically to a flag polypeptide.  
     
     
         34 . The kit of  claim 33  wherein the solid phase comprises a well of a microtiter plate.  
     
     
         35 . The kit of  claim 33  further comprising a labeled anti-phosphotyrosine antibody.  
     
     
         36 . The kit of  claim 35  wherein the label comprises an enzyme.  
     
     
         37 . The kit of  claim 33  further comprising a cell transformed with a nucleic acid encoding a receptor construct.  
     
     
         38 . An assay for measuring phosphorylation of a kinase comprising the steps of: 
 (a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein the cells comprise a kinase construct comprising a flag polypeptide and the kinase;    (b) exposing the adhering cells to an analyte;    (c) solubilizing the adhering cells, thereby releasing cell lysate therefrom;    (d) coating a second solid phase with a capture agent which binds specifically to the flag polypeptide so that the capture agent adheres to the second solid phase;    (e) exposing the adhering capture agent to the cell lysate obtained in step (c) so that the kinase construct adheres to the second solid phase;    (f) washing the second solid phase so as to remove unbound cell lysate;    (g) exposing the adhering kinase construct to an antibody which identifies phosphorylated residues in the kinase construct; and    (h) measuring binding of the antibody to the adhering kinase construct.    
     
     
         39 . The assay of  claim 38  wherein the kinase is a receptor.  
     
     
         40 . The assay of  claim 38  wherein the kinase is a serine-threonine kinase.  
     
     
         41 . The assay of  claim 38  which measures phosphatase activity.  
     
     
         42 . The assay of claim  41  wherein the cells further comprise a phosphatase and the assay further comprises the step of exposing the eukaryotic cells to a phosphatase inhibitor prior to step (c).  
     
     
         43 . The assay of claim  41  which further comprises the steps in between steps (f) and (g) of exposing the adhering kinase construct to a phosphatase and then washing the second solid phase so as to remove unbound phosphatase.