Human platelet-derived growth factor receptors

Abstract

DNA sequences encoding human platelet-derived growth factor receptors (hPDGF-R), and expression constructs comprising sequences which encode a receptor that can be secreted or incorporated into the membrane of a mammalian cell. Peptide fragments with functions equivalent to the wild-type receptor, conferring a PDGF-sensitive mitogenic response on cells lacking the receptor are provided. The constructs can be used for enhancing PDGF response of cells, determining the regions involved in transducing the signal in response to PDGF binding, providing mutated analogs and evaluating drugs for their physiologic activity. Soluble fragments comprising PDGF receptor sequences are also provided, including important intracellular kinase insert sequences which interact with intracellular proteins.

1 . A purified and isolated recombinant nucleic acid of less than about 50 kbp comprising at least about 24 contiguous nucleotides which encode a human platelet-derived growth factor receptor (hPDGF-R) polypeptide segment.  
     
     
         2 . A nucleic acid of  claim 1 , wherein said segment is selected from the group consisting of: 
 a) a segment which comprises a phosphorylation site;    b) a segment which is capable of binding to PDGF;    c) a segment which is a substrate for phosphorylation; and    d) a segment which is capable of binding to a PI3 kinase.    
     
     
         3 . A cell transformed with a nucleic acid of  claim 1 , and wherein said cell is a mammalian cell.  
     
     
         4 . A nucleic acid of  claim 1 , wherein said nucleotides encoding said segment are operably linked to a promoter.  
     
     
         5 . A nucleic acid of  claim 1 , further encoding a heterologous polypeptide which is fused to said hPDGF-R segment.  
     
     
         6 . A composition comprising a combination of: a) the nucleic acid of  claim 1;  and b) a non-fungal glycosylation enzyme capable of glycosylating said fragment when expressed.  
     
     
         7 . A method for evaluating the ability of a compound to function as a hPDGF-R agonist or antagonist comprising the step of comparing the amount of a PDGF-induced response in a cell of  claim 3  with the response from a control cell, and wherein said PDGF-induced response is compared by measuring synthesis of DNA in a cell after contacting said cell with PDGF.  
     
     
         8 . A substantially pure hPDGF-R polypeptide fragment of at least about twenty amino acids having platelet-derived growth factor (PDGF) receptor binding activity or tyrosine kinase activity.  
     
     
         9 . A composition comprising an unglycosylated hPDGF-R fragment.  
     
     
         10 . A composition comprising a hPDGF-R fragment, which exhibits a glycosylation pattern which is non-fungal and non-human.  
     
     
         11 . A method for introducing a hPDGF-R activity to a cell, said method comprising the step of introducing a hPDGF-R protein fragment of  claim 8  to said cell, wherein the hPDGF-R protein fragment comprises at least about five hundred daltons.  
     
     
         12 . A method for assaying the presence of a ligand for a PDGF receptor in a sample, comprising the steps of: combining said sample with the hPDGF receptor fragment of  claim 8 , wherein the hPDGF-R fragment comprises the ligand binding site; and detecting binding between said ligand and said hPDGF receptor ligand binding site.  
     
     
         13 . An isolated hPDGF-R polypeptide of less than about 200 amino acids comprising a receptor kinase insert region.  
     
     
         14 . The isolated polypeptide of  claim 13 , wherein said polypeptide has a phosphorylated amino acid residue.  
     
     
         15 . An isolated polypeptide of  claim 13 , with a pharmaceutically acceptable carrier.  
     
     
         16 . A method for modulating the biological activity of a first protein which binds to a phosphorylated polypeptide of  claim 14 , said method comprising a step of: adding to said first protein a peptide analogue of said polypeptide, wherein said analogue is capable of inhibiting the binding of said first protein to said polypeptide.  
     
     
         17 . A method of selecting a molecule capable of inhibiting binding of a protein which binds to the phosphorylated polypeptide of  claim 14 , comprising the steps of: contacting said protein with said phosphorylated polypeptide in the presence of said molecule in a first analysis; contacting said protein with said phosphorylated polypeptide in the absence of said molecule in a second analysis; and comparing said analyses to determine the effect of said molecule on said binding.  
     
     
         18 . A method of purifying, from a sample, a protein capable of binding to the hPDGF-R polypeptide of  claim 13 , comprising the step of: contacting said sample with an analogue of a phosphorylated polypeptide substantially homologous to the hPDGF-R polypeptide, thereby allowing said protein to bind specifically to said phosphorylated polypeptide.  
     
     
         19 . A method of isolating a nucleic acid encoding a protein capable of binding to a PDGF receptor, comprising the steps of: combining a labeled polypeptide of  claim 14  with cells expressing various proteins, thereby labeling those cells which express said nucleic acid to produce a protein which binds said polypeptide, and isolating those cells which have been labeled.  
     
     
         20 . A method of  claim 19 , wherein said protein capable of binding a PDGF receptor is PI3 kinase or c-fins.