Interspecies molecular chimeras of kit help define the binding site of the stem cell factor.
The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.
Lev
S
S
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Blechman
J
J
Nishikawa
S
S
Givol
D
D
Yarden
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D016298:Hematopoietic Cell Growth Factors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D019089:Stem Cell Factor; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D016298:Hematopoietic Cell Growth Factors; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit