Oligomerized receptors which affect pathways regulated by transmembrane ligands for elk-related receptor tyrosine kinases

Abstract

A method of modulating the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand, comprising forming a complex between a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the kinase, and a transmembrane ligand expressed on the cell, thereby affecting a pathway in the cell which is regulated by the transmembrane ligand.

We claim:  
     
         1 . A method of modulating the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand comprising forming a complex between a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase, and the transmembrane ligand expressed on the cell, thereby modulating the biological activity of the transmembrane ligand.  
     
     
         2 . A method of affecting a pathway regulated by a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand, comprising forming a complex between a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase, and a transmembrane ligand expressed on the cell, thereby affecting or modulating a pathway in the cell which is regulated by the transmembrane ligand.  
     
     
         3 . A method for evaluating a substance for its ability to modulate the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand comprising the steps of: 
 (a) contacting an oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase; a transmembrane ligand which binds to the Elk-related receptor tyrosine kinase to form a receptor-ligand complex, wherein the transmembrane ligand is a transmembrane ligand for an Elk-related receptor tyrosine kinase expressed on a cell; and, a test substance, under conditions which permit the formation of receptor-ligand complexes;    (b) assaying for receptor-ligand complexes, free Elk-related receptor tyrosine kinase, or non-complexed transmembrane ligand, or for activation of the transmembrane ligand; and    (c) comparing to a control to determine if the substance inhibits or enhances the binding of the Elk-related receptor tyrosine kinase and transmembrane ligand, and thereby modulates the biological activity of the transmembrane ligand.    
     
     
         4 . A method for identifying a substance which affects or modulates a pathway regulated by a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand, comprising the steps of: 
 (a) contacting an oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase; a transmembrane ligand which binds to the Elk-related receptor tyrosine kinase to form receptor-ligand complexes which activate a pathway regulated by a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand; and a test substance under conditions which permit the formation of receptor-ligand complexes;    (b) assaying for receptor-ligand complexes, free Elk-related receptor tyrosine kinase, or non-complexed transmembrane ligand, or for activation of the transmembrane ligand; and    (c) comparing to a control to determine if the substance inhibits or enhances the binding of the Elk related receptor tyrosine kinase and transmembrane ligand and thereby affects or modulates the pathway.    
     
     
         5 . A method for evaluating a substance for its ability to inhibit or enhance the interaction of an oligomerized Elk-related receptor tyrosine kinase, or an isoform, or an extracellular domain of the Elk-related receptor tyrosine kinase, and a transmembrane ligand for an Elk-related receptor tyrosine kinase expressed on a cell comprising: 
 (a) providing a reporter gene operably linked to a DNA binding site for a transcriptional activator;    (b) providing a first hybrid protein comprising the transmembrane ligand in polypeptide linkage to a DNA binding domain of the transcriptional activator;    (c) providing a second hybrid protein comprising an oligomerized Elk-related receptor tyrosine kinase or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase in polypeptide linkage to an activation domain of the transcriptional activator; under conditions where the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase can bind and thereby reconstitute the transcriptional activator which induces transcription of the reporter gene;    (d) administering a test substance; and    (e) monitoring expression of the reporter gene, wherein a decrease in expression is an indication that the substance inhibits the interaction of the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase, and an increase in expression is an indication that the substance enhances the interaction of the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase.    
     
     
         6 . A method for evaluating a substance for its ability to inhibit or enhance the interaction of an oligomerized Elk-related receptor tyrosine kinase, or an isoform, or an extracellular domain of the Elk-related receptor tyrosine kinase, and a transmembrane ligand for an Elk-related receptor tyrosine kinase expressed on a cell comprising: 
 (a) providing a reporter gene operably linked to a DNA binding site for a transcriptional activator, (b) providing a first hybrid protein comprising an oligomerized Elk-related receptor tyrosine kinase or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase in polypeptide linkage to a DNA binding domain of the transcriptional activator;    (c) providing a second hybrid protein comprising a transmembrane ligand in polypeptide linkage to an activation domain of the transcriptional activator; under conditions where the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase can bind and thereby reconstitute the transcriptional activator which induces transcription of the reporter gene;    (d) administering a test substance; and    (e) monitoring expression of the reporter gene, wherein a decrease in expression is an indication that the substance inhibits the interaction of the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase, and an increase in expression is an indication that the substance enhances the interaction of the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase.    
     
     
         7 . A method as claimed in any one of  claims 1  to  6 , wherein the transmembrane ligand is Elk-L/LERK2/Efl-3/Cek5-L, hHtk-L/ELF-2/Lerk5, or hElk-L3/Efl-6.  
     
     
         8 . A method as claimed in any one of  claims 1  to  7 , wherein the Elk-related receptor tyrosine kinase is Nuk, Hek5, Erk, Sek3, Cek5 , Elk, Cek6a, xEK, Hek2, Sek4, Cek10 , Htk, or Myk1.  
     
     
         9 . A method as claimed in any one of  claims 1  to  6  wherein the transmembrane ligand is Elk-L and the Elk-related receptor tyrosine kinase is Elk or Nuk.  
     
     
         10 . A method as claimed in any one of  claims 1  to  6  wherein the transmembrane ligand is Htk-L and the Elk-related receptor tyrosine kinase is Elk or Nuk.  
     
     
         11 . An antibody preparation which specifically binds to a receptor-ligand complex comprising an oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the kinase, and a transmembrane ligand for an Elk-related receptor tyrosine kinase.  
     
     
         12 . Use of a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain thereof, in the preparation of a medicament for modulating the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand.  
     
     
         13 . Use of a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain thereof, in the preparation of a medicament for modulating neuronal development or regeneration in a subject.  
     
     
         14 . Use of a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain thereof, in the preparation of a medicament for modulating axonogenesis in a subject .  
     
     
         15 . A use as claimed in any one of  claims 12  to  14 , wherein the transmembrane ligand is Elk-L/LERK2/Efl-3/Cek5-L, hHtk-L/ELF-2/Lerk5, or hElk-L3/Efl6.  
     
     
         16 . A use as claimed in any one of  claims 12  to  15 , wherein the Elk-related receptor tyrosine kinase is Nuk, Hek5, Erk, Sek3, Cek5, Elk, Cek6a, xEK, Hek2, Sek4, Cek10, Htk, or Myk1.  
     
     
         17 . A use as claimed in any one of  claims 12  to  14 , wherein the transmembrane ligand is Elk-L and the Elk-related receptor tyrosine kinase is Elk or Nuk.  
     
     
         18 . Use of a substance identified in accordance with a method as claimed in any one of  claims 1  to  10  in the preparation of a medicament for modulating the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand.  
     
     
         19 . A pharmaceutical composition which comprises a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain thereof, in an amount effective to stimulate or inhibit neuronal development or regeneration and a pharmaceutically acceptable carrier, diluent or excipient.