A recombinant ectodomain of the receptor for the stem cell factor (SCF) retains ligand-induced receptor dimerization and antagonizes SCF-stimulated cellular responses.
The stem cell factor (SCF) is a polypeptide ligand that is essential for the development of germ cells, hematopoietic progenitor cells, and melanocyte precursors. It binds to a tyrosine kinase membrane receptor that is encoded by the c-kit proto-oncogene. We have constructed an expression vector that directs the synthesis of the entire extracellular ligand-binding domain of the Kit/SCF receptor. When expressed and amplified in Chinese hamster ovary cells, a secreted 90-kDa glycoprotein could be harvested from the growth medium of the cells in a soluble form. This extracellular portion of the Kit/SCF receptor, denoted Kit-X, was recognized by antibodies specific to the SCF receptor; and when injected into animals, it raised antibodies that were reactive with the complete membrane form of the receptor. Direct binding and covalent cross-linking of radiolabeled SCF showed that Kit-X fully retained high affinity ligand binding and also underwent efficient dimerization in the presence of the ligand. The capacity of Kit-X to act as an antagonist of SCF was assayed on cultured cells that overexpress the receptor. Simultaneous addition of SCF and Kit-X to these cells resulted in a stoichiometric inhibition of SCF binding and a consequent decrease in autophosphorylation of the SCF receptor on tyrosine residues. The inhibition extended to later SCF-mediated responses, including the association of the receptor with phosphatidylinositol 3'-kinase and coupling to the Raf1 protein kinase. These results indicate that the recombinant ectodomain of the Kit-SCF receptor can be used as a specific antagonist of SCF actions and may enable detailed molecular analysis of ligand-receptor interactions.
Lev
S
S
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Yarden
Y
Y
Givol
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004247:DNA; D005822:Genetic Vectors; D016298:Hematopoietic Cell Growth Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019089:Stem Cell Factor; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D003432:Cross-Linking Reagents; D016298:Hematopoietic Cell Growth Factors; D008024:Ligands; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D014443:Tyrosine; D004247:DNA; D019009:Proto-Oncogene Proteins c-kit