Interaction of macrophage-stimulating protein with its receptor. Residues critical for beta chain binding and evidence for independent alpha chain binding.
Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors. Each one is a heterodimeric protein comprising a disulfide-linked alpha chain and a serine protease-like beta chain. Despite structural similarities between MSP and HGF, the primary receptor binding site is located on the alpha chain of HGF/SF but on the beta chain of MSP. To obtain insight into the structural basis for MSP beta chain binding, beta chain structure was modeled from coordinates of an existing model of the HGF beta chain. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn(682)/Glu(648) interacting pair, leaving a shallow cavity for possible beta chain interaction with the receptor. Mutants in this region were created, and their binding characteristics were determined. A double mutation of Asn(682)/Glu(648) caused diminished binding of the beta chain to the MSP receptor, and a single mutation of neighboring Arg(683) completely abolished binding. Thus, this region of the molecule is critical for binding. We also found that at equimolar concentrations of free alpha and beta chains, alpha chain binding to receptor was detectable, at levels considerably lower than beta chain binding. The EC(50) values determined by quantitative enzyme-linked immunosorbent assay are 0.25 and 16.9 nM for beta and alpha chain, respectively. The data suggest that MSP has two independent binding sites with high and low affinities located in beta and alpha chain, respectively, and that the two sites together mediate receptor dimerization and subsequent activation.
Danilkovitch
A
A
Laboratory of Immunobiology, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, Maryland 21702, USA. danilkovitch@mail.ncifcrf.gov
Miller
M
M
Leonard
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary; D004285:Dogs; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017228:Hepatocyte Growth Factor; D008969:Molecular Sequence Data; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism
D018076:DNA, Complementary; D006133:Growth Substances; D011518:Proto-Oncogene Proteins; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met; D012697:Serine Endopeptidases