Glial cell line-derived neurotrophic factor receptor

Abstract

The present invention relates to glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophin that exhibits a broad spectrum of biological activities on a variety of cell types from both the central and peripheral nervous systems. The present invention involves the cloning and characterization of a high affinity receptor for GDNF. This molecule has been named GDNF receptor (GDNFR) since it is the first known component of a receptor system. Nucleic acid and amino acid sequences are described for GDNFR protein products. A hydrophobic domain with the features of a signal peptide is found at the amino terminus, while a second hydrophobic domain at the carboxy terminus is involved in the linkage of the receptor to the cell membrane. The lack of a transmembrane domain and cytoplasmic region indicates that GDNFR requires one or more accessory molecules in order to mediate transmembrane signaling. GDNFR mRNA is widely distributed in both nervous system and non-neural tissues, consistent with the similar distribution found for GDNF.

What is claimed is:  
     
         1 . An isolated and purified protein comprising an amino acid sequence as depicted in FIGS.  2  or  4  (SEQ ID NO: 2 or 4) and analogs thereof wherein the protein is capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and thereby mediating cell response to GDNF.  
     
     
         2 . A protein of  claim 1  comprising the amino acid sequence as depicted in FIG. 2 (SEQ ID NO: 2).  
     
     
         3 . A protein of  claim 1  comprising the amino acid sequence as depicted in FIG. 4 (SEQ ID NO:4).  
     
     
         4 . A protein of  claim 1  comprising the amino acid sequence Ser 18  through Pro 446  as depicted in FIG. 2 (SEQ ID NO:2).  
     
     
         5 . A protein of  claim 1  comprising the amino acid sequence Asp 25  through Leu 447  as depicted in FIG. 2 (SEQ ID NO:2).  
     
     
         6 . A protein of  claim 1  comprising the amino acid sequence Cys 29  through Cys 442  as depicted in FIG. 2 (SEQ ID NO:2).  
     
     
         7 . A protein of  claim 1  comprising the amino acid sequence Ala 19  through Val 450  as depicted in FIG. 4 (SEQ ID NO:4).  
     
     
         8 . A protein of  claim 1  comprising the amino acid sequence Cys 29  through Cys 443  as depicted in FIG. 4 (SEQ ID NO:4).  
     
     
         9 . A protein of  claim 1  which is glycosylated.  
     
     
         10 . A protein of  claim 1  which is non-glycosylated.  
     
     
         11 . A protein of  claims 1  to  10  which is produced by recombinant technology or chemical synthesis.  
     
     
         12 . A pharmaceutical composition comprising a protein as claimed in any one of  claims 1  to  10  in combination with a pharmaceutically acceptable carrier.  
     
     
         13 . An isolated nucleic acid sequence encoding a neurotrophic factor receptor protein comprising an amino acid sequence as claimed in any one of  claims 1  to  8 .  
     
     
         14 . An isolated nucleic acid sequence encoding a neurotrophic factor receptor protein comprising an amino acid sequence as depicted in FIGS.  2  or  4  (SEQ ID NO: 2 or 4) and analogs thereof wherein the protein is capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and thereby mediating cell response to GDNF.  
     
     
         15 . A nucleic acid sequence of  claim 14  encoding a neurotrophic factor receptor protein comprising the amino acid sequence as depicted in FIG. 2 (SEQ ID NO: 2).  
     
     
         16 . A nucleic acid sequence of  claim 14  encoding a neurotrophic factor receptor protein comprising the amino acid sequence as depicted in FIG. 4 (SEQ ID NO:4).  
     
     
         17 . An isolated nucleic acid sequence comprising: 
 (a) a sequence set forth in FIG. 1 (SEQ ID NO: 1) comprising nucleotides encoding Met 1  through Ser 465  or FIG. 3 (SEQ ID NO: 3) comprising nucleotides encoding Met 1  through Ser 468 , wherein said sequence encodes a neurotrophic factor receptor protein (GDNFR) capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and thereby mediating cell response to GDNF;    (b) a nucleic acid sequence which (1) hybridizes to a complementary sequence of (a) and (2) encodes an amino acid sequence with GDNFR activity; and    (c) a nucleic acid sequence which but for the degeneracy of the genetic code would hybridize to a complementary sequence of (a) and (2) encodes an amino acid sequence with GDNFR activity.    
     
     
         18 . A vector comprising a nucleic acid sequence according to any of  claims 14  to  17  operatively linked to one or more operational elements capable of effecting the amplification or expression of said nucleic acid sequence.  
     
     
         19 . A vector comprising a nucleic acid sequence encoding a neurotrophic factor receptor protein comprising the amino acid sequence as depicted in FIGS.  2  or  4  (SEQ ID NO: 2 or 4) operatively linked to one or more operational elements capable of effecting the amplification or expression of said nucleic acid sequence.  
     
     
         20 . A host cell transformed or transfected with the vector of  claim 18 .  
     
     
         21 . A host cell transformed or transfected with the vector of  claim 19 .  
     
     
         22 . A host cell of  claim 20  selected from the group consisting of mammalian cells and bacterial cells.  
     
     
         23 . A host cell of  claim 22  which is a COS-7 cell or  E. coli.    
     
     
         24 . A host cell of  claim 20  wherein said cell is suitable for human implantation and wherein said cell expresses and secretes said neurotrophic factor receptor.  
     
     
         25 . A host cell of  claim 21  wherein said cell is suitable for human implantation and wherein said cell expresses and secretes said neurotrophic factor receptor.  
     
     
         26 . A host cell of  claim 20  wherein said cell is transformed or transfected ex vivo.  
     
     
         27 . A host cell of  claim 20  wherein said cell is enclosed in a semipermeable membrane suitable for human implantation.  
     
     
         28 . A method for the production of a neurotrophic factor receptor protein comprising the steps of: 
 (a) culturing a host cell, containing a nucleic acid sequence encoding a neurotrophic factor receptor protein comprising an amino acid sequence as depicted in FIGS.  2  or  4  (SEQ ID NO: 2 or 4) and analogs thereof wherein the protein is capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and thereby mediating cell response to GDNF, under conditions suitable for the expression of said neurotrophic factor receptor protein by said host cell; and    (b) optionally, isolating said neurotrophic factor receptor protein expressed by said host cell.    
     
     
         29 . A method of  claim 28 , wherein said nucleic acid sequence encodes a neurotrophic factor receptor protein comprising the amino acid sequence as depicted in FIG. 2 (SEQ ID NO:2).  
     
     
         30 . A method of  claim 28 , wherein said nucleic acid sequence encodes a neurotrophic factor receptor protein comprising the amino acid sequence as depicted in FIG. 4 (SEQ ID NO:4).  
     
     
         31 . A method for the production of a neurotrophic factor receptor protein comprising the steps of: 
 (a) culturing a host cell transformed or transfected with a nucleic acid sequence according to  claim 17  under conditions suitable for the expression of said neurotrophic factor receptor protein by said host cell; and    (b) optionally, isolating said neurotrophic factor receptor protein expressed by said host cell.    
     
     
         32 . A method of  claim 28  or  31 , further comprising the step of refolding the isolated neurotrophic factor receptor.  
     
     
         33 . A method of  claim 28  or  31 , wherein said host cell is a prokaryotic cell.  
     
     
         34 . A method of  claim 28  or  31 , wherein said host cell is a eukaryotic cell.  
     
     
         35 . A substantially purified neurotrophic factor receptor protein prepared according to the method of any of  claims 28  to  31 .  
     
     
         36 . The use of the neurotrophic factor receptor protein of  claim 1  for the manufacture of a pharmaceutical composition.  
     
     
         37 . A method of treating improperly functioning dopaminergic nerve cells by administering a neurotrophic factor receptor protein of  claim 1 .  
     
     
         38 . A method of treating Parkinson's disease by administering a neurotrophic factor receptor protein of  claim 1 .  
     
     
         39 . A method of treating Alzheimer's disease by administering a neurotrophic factor receptor protein of  claim 1 .  
     
     
         40 . A method of treating amyotrophic lateral sclerosis by administering a neurotrophic protein of  claim 1 .  
     
     
         41 . An antibody that binds to a neurotrophic factor receptor protein comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.  
     
     
         42 . The antibody of  claim 41  wherein said antibody is a monoclonal antibody.  
     
     
         43 . The antibody of  claim 41  wherein said antibody is a polyclonal antibody.  
     
     
         44 . An antibody produced by immunizing an animal with a neurotrophic factor receptor protein comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.  
     
     
         45 . A hybridoma that produces a monoclonal antibody that binds to a neurotrophic factor receptor protein comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.  
     
     
         46 . A device for treating nerve damage, comprising: 
 (a) a semipermeable membrane suitable for implantation; and    (b) cells encapsulated within said membrane, wherein said cells secrete a neurotrophic factor receptor protein according to  claim 1;     said membrane being permeable to the neurotrophic factor receptor protein and impermeable to materials detrimental to said cells.    
     
     
         47 . The device of  claim 46 , wherein said cells are naturally occurring cells that secrete said neurotrophic factor receptor protein.  
     
     
         48 . The device of  claim 46 , wherein said cells have been modified to secrete said neurotrophic factor receptor protein by means of a nucleic acid sequence comprising: 
 (a) a sequence set forth in FIG. 1 (SEQ ID NO.: 1) comprising nucleotides encoding Met 1  through Ser 465  or FIG. 3 (SEQ ID NO: 3) comprising nucleotides encoding Met 1  through Ser 468  encoding a neurotrophic factor receptor protein (GDNFR) capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and mediating cell response to GDNF;    (b) a nucleic acid sequence which (1) hybridizes to a complementary sequence of (a) and (2) encodes an amino acid sequence with GDNFR activity; and    (c) a nucleic acid sequence which but for the degeneracy of the genetic code would hybridize to a complementary sequence of (a) and (2) encodes an amino acid sequence with GDNFR activity.    
     
     
         49 . An assay device for analyzing a test sample for the presence of glial cell line-derived neurotrophic factor, comprising: a solid phase containing or coated with a GDNFR protein, wherein said GDNFR protein reacts with GDNF present in the test sample and produces a detectable reaction product indicative of the presence of GDNF.  
     
     
         50 . A method for analyzing a test sample for the presence of glial cell line-derived neurotrophic factor, comprising: contacting the sample to an assay reagent comprising GDNFR protein, wherein said GDNFR protein reacts with GDNF present in the test sample and produces a detectable reaction product indicative of the presence of GDNF.