Soluble c-kit proteins and antireceptor monoclonal antibodies confine the binding site of the stem cell factor.
The binding of the stem cell factor (SCF) to the c-kit-encoded receptor tyrosine kinase stimulates a variety of biochemical responses that culminate in cellular proliferation, migration, or survival. The extracellular domain of p145kit consists of five immunoglobulin-like domains. To confine the ligand binding site to this portion of the receptor we generated a panel of murine monoclonal antibodies (mAbs) to the Kit protein and identified two mAbs that efficiently displaced receptor-bound SCF and also inhibited proliferation of SCF-dependent human megakaryocytes. To map the epitopes of these mAbs we constructed and expressed soluble portions of the extracellular domain of Kit, which included either the two amino-terminal Ig-like domains (denoted Kit 1-2), three Ig-like domains (Kit 1-2-3), or the entire extracellular portion (Kit-X). All three recombinant proteins were recognized by the ligand inhibitory mAbs, suggesting that the SCF binding site resides in the amino-terminal half of the ecto-domain. Consistent with this conclusion, all of the soluble proteins inhibited SCF binding to Kit-expressing cells, and they also underwent specific covalent cross-linking to the radiolabeled ligand. However, whereas Kit 1-2-3 and Kit-X displayed comparable ligand affinities, deletion of the third Ig-like domain, in Kit 1-2, involved significant reduction in SCF binding. Hence, the binding site of SCF probably includes Ig-like domains 1 and 2, but structural determinants distal to this portion may also participate in ligand recognition.
Blechman
J M
JM
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Lev
S
S
Brizzi
M F
MF
Leitner
O
O
Pegoraro
L
L
Givol
D
D
Yarden
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D016298:Hematopoietic Cell Growth Factors / Q000378:metabolism*; D009154:Mutation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit; D016184:Receptors, Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D019089:Stem Cell Factor
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D016298:Hematopoietic Cell Growth Factors; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit