Method of use of sonic hedgehog protein as a ligand for patched

Abstract

The invention relates to hedgehog proteins that are involved in signaling developmental processes. The proteins of the invention modulate differentiation of neural plate. Also provided are methods for identifying compounds that modulate early events in development and more particularly to methods to identify compounds that modulate differentiation of neural plate using hedgehog proteins as ligands for the patched receptor.

1 . An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:1 wherein alanine is substituted for the amino acid residue at a position selected from 51, 52, 56, 75, 90, 76, 81, 105, 116, 132, 135, 138, 168, 177, 189 and 195, and any combination thereof.  
     
     
         2 . An isolated protein comprising the amino acid sequence set forth in a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:12.  
     
     
         3 - 46 . (canceled)  
     
     
         47 . The isolated protein of  claim 1 , wherein at least one thiol-containing amino acid is substituted at a position corresponding to position 51, 52, 56, 75, 90, 76, 81, 105, 116, 132, 135, 138, 168, 177, 189 and 195, and any combination thereof, and wherein the protein is modified by attachment to a maleimide-linked moiety.  
     
     
         48 . The isolated protein of  claim 47 , wherein the thiol-containing amino acid is cysteine or methionine.  
     
     
         49 . The isolated protein of  claim 47 , wherein the maleimide-linked moiety is a polyethylene glycol (PEG) moiety.  
     
     
         50 . The isolated protein of  claim 2 , wherein at least one thiol-containing amino acid is substituted at a position corresponding to position 51, 52, 56, 75, 90, 76, 81, 105, 116, 132, 135, 138, 168, 177, 189 and 195, and any combination thereof, and wherein the protein is modified by attachment to a maleimide-linked moiety.  
     
     
         51 . The isolated protein of  claim 50 , wherein the thiol-containing amino acid is cysteine or methionine.  
     
     
         52 . The isolated protein of  claim 50 , wherein the maleimide-linked moiety is a polyethylene glycol (PEG) moiety.  
     
     
         53 . An isolated protein comprising the amino acid sequence set forth in a sequence selected from SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.  
     
     
         54 . The isolated protein of  claim 53 , wherein at least one thiol-containing amino acid is substituted at a position corresponding to position 51, 52, 56, 75, 90, 76, 81, 105, 116, 132, 135, 138, 168, 177, 189 and 195, and any combination thereof, and wherein the protein is modified by attachment to a maleimide-linked moiety.  
     
     
         55 . The isolated protein of  claim 54 , wherein the thiol-containing amino acid is cysteine or methionine.  
     
     
         56 . The isolated protein of  claim 54 , wherein the maleimide-linked moiety is a polyethylene glycol (PEG) moiety.