Soluble c-kit proteins and antireceptor monoclonal antibodies confine the binding site of the stem cell factor
| Autor: | J M, Blechman, S, Lev, M F, Brizzi, O, Leitner, L, Pegoraro, D, Givol, Y, Yarden |
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| Rok vydání: | 1993 |
| Předmět: |
Stem Cell Factor
Binding Sites Antibodies Monoclonal CHO Cells Protein-Tyrosine Kinases Hematopoietic Cell Growth Factors Recombinant Proteins Proto-Oncogene Proteins c-kit Cross-Linking Reagents Solubility Cricetinae Proto-Oncogene Proteins Mutation Receptors Colony-Stimulating Factor Animals Sequence Deletion |
| Zdroj: | The Journal of biological chemistry. 268(6) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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