Glial cell line-derived neurotrophic factor receptors

Abstract

Receptors for Glial Cell Line-Derived Neurotrophic Factor (GDNF), their cellular expression, isolation, biochemical characterization, and sequences are disclosed. c-RET is disclosed as one receptor for GDNF; additional novel receptors are also disclosed. The preparation of monoclonal antibodies directed against GDNF is also disclosed.

What is claimed is:  
     
         1 . An isolated receptor which binds glial cell lined-derived neurotrophic factor (GDNF), said receptor comprising at least one polypeptide having a molecular weight selected from the group consisting of polypeptides of about 55 kD, 70 kD, 135 kD, and 300 kD molecular weight, as determined by SDS-PAGE on 4-20% gradient gels.  
     
     
         2 . A competitive assay for identifying compounds which bind to GDNF receptors comprising 
 a) incubating said compounds with cells which express c-RET receptors in the presence of an excess of labeled GDNF;    b) measuring the amount of labeled GDNF bound to said cells; and    c) comparing amount labeled GDNF bound to said cells to that of controls not incubated with said compounds.    
     
     
         3 . The method of  claim 2  wherein the cells are selected from the group consisting of NB2/a, MN-1, and PC12 cells.  
     
     
         4 . The method of  claim 2  wherein the labeled GDNF is  125 I-GDNF.  
     
     
         5 . A competitive assay for identifying compounds which bind to isolated GDNF receptors comprising 
 a) incubating said compounds with isolated c-RET receptors in the presence of an excess of labeled GDNF;    b) measuring the amount of labeled GDNF bound to said receptors; and    c) comparing amount labeled GDNF bound to said receptors to that of controls not incubated with said compounds.    
     
     
         6 . The method of  claim 5  wherein the receptors are polypeptides which bind GDNF selected from the group consisting of polypeptides about 55 kD, 70 kD, 135 kD, 155 kD, and 300 kD molecular weight.  
     
     
         7 . The method of  claim 6  wherein the polypeptide is about 155 kD molecular weight.  
     
     
         8 . The method of  claim 5  wherein the isolated receptor is c-RET.  
     
     
         9 . The method of  claim 5  wherein the labeled GDNF is  125 I-GDNF.  
     
     
         10 . A method for identifying compounds which are GDNF homologs comprising 
 a) incubating said compounds with cells which express c-RET receptors; and    b) determining whether said compound effects tyrosine phosphorylation.    
     
     
         11 . The method of  claim 10  wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.  
     
     
         12 . A method for identifying compounds which are GDNF homologs comprising 
 a) incubating said compounds with cells which express c-RET receptors; and    b) determining whether said compounds effect an increase in c-fos mRNA levels.    
     
     
         13 . The method of  claim 12  wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.  
     
     
         14 . A method for identifying compounds which are GDNF homologs comprising 
 a) incubating said compounds with cells which express c-RET receptors under non-permissive conditions for said cells; and    b) determining the number of surviving cells as compared to controls not incubated with said compounds.    
     
     
         15 . The method of  claim 14  wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.  
     
     
         16 . A method for identifying compounds which are GDNF analogs comprising 
 a) incubating said compounds with cells which express c-RET receptors in the presence of concentrations of GDNF effective for phosphorylating tyrosine; and    b) determining whether said compounds effect a decrease in the tyrosine phosphorylation as compared with controls not incubated with said compounds.    
     
     
         17 . The method of  claim 16  wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.  
     
     
         18 . A method for identifying compounds which are GDNF analogs comprising 
 a) incubating said compounds with cells which express c-RET receptors in the presence of concentrations of GDNF effective for increasing c-fos mRNA levels; and    b) determining whether said compounds effect a decrease in c-fos mRNA levels as compared with controls not incubated with said compounds.    
     
     
         19 . The method of  claim 18  wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.  
     
     
         20 . A method for identifying compounds which are GDNF analogs comprising 
 a) incubating said compounds with cells which express c-RET receptors under non-permissive conditions for said cells in the presence of amount of GDNF effective for cell survival; and    b) determining the number of surviving cells as compared with controls not incubated with said compounds.    
     
     
         21 . The method of  claim 20  wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.  
     
     
         22 . Isolated GDNFR-β comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         23 . Isolated GDNFR-β comprising the amino acid sequence of SEQ ID NO:9.  
     
     
         24 . A compound comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         25 . A compound comprising the amino acid sequence of SEQ ID NO:9.  
     
     
         26 . An ioslated nucleic acid having the sequence of SEQ ID No:5.  
     
     
         27 . An ioslated nucleic acid having the sequence of SEQ ID No: 10.