Abstract
Our previous studies have suggested that a derivative of hepatocyte growth factor (HGF), HGF/NK2, containing the coding sequences for the N-terminal hairpin and first two kringle domains, is sufficient to mediate high affinity binding to the HGF receptor. Here, we wished to test directly whether HGF/NK1 (N-terminal hairpin and first kringle domains) could bind the receptor and/or mediate receptor signaling. HGF/NK1 was expressed in Escherichia coli and purified to homogeneity using heparin-affinity and fast protein liquid cation-exchange chromatography. Biological characterization of HGF/NK1 showed that it can compete for binding to the HGF receptor on human lung carcinoma A549 cells and to a soluble form of the HGF receptor. HGF/NK1 is inefficient at promoting autophosphorylation of the HGF receptor, although some activity was detected at very high concentrations. HGF/NK1 fails to exhibit mitogenic properties even at very high concentrations. However, HGF/NK1 can act as a potent competitive antagonist in this assay. Our data demonstrate directly that a receptor binding determinant of HGF is located within the N-terminal 32-212 residues of HGF. HGF/NK1 will serve as a powerful tool for (i) generating neutralizing antibodies, (ii) in determining x-ray crystallographic and nuclear magnetic resonance structures, and (iii) for in vivo studies as an HGF antagonist.
Highlights
Our previous studies have suggested that a deriva- teolytic activity, presumably because residues within the cattive ofhepatocytegrowthfactor(HGF),HGF/NK2, alytic triad are notconserved (Nakamura et al, 1989)
TheHGF receptor is the product of the c-Met protohairpin and first two kringle domains, is sufficient to oncogene, amembrane-spanning tyrosine kinase receptor mediatehigh affinity bindingtothe HGF receptor. (Bottaro et al, 1991; Naldini et al, 1991)
The HGF receptor is autophosphorylated on tyrosine residues of the 145-kDa@-subunitupon HGF binding (Naldini et al, 1991,Lokker et al, 1992).We haveexpressed a chimeric protein containingthe extracellular domain of the HGF receptor fused to the constantregion of a human IgG heavy chain
Summary
Our previous studies have suggested that a deriva- teolytic activity, presumably because residues within the cattive ofhepatocytegrowthfactor(HGF),HGF/NK2, alytic triad are notconserved (Nakamura et al, 1989). Iological characterizationof HGF/NKl showed thatit can compete for binding to the HGF receptor on human lung carcinomaA649 cells and toa soluble form of thHeGF receptor. We wished to determine whether HGF/NKl itself might be sufficient for HGF receptor binding. ’The abbreviations used are: HGF, hepatocyte growth factor; FPLC, fast protein liquid chromatography; HGF/NKl, N-terminal hairpin and kringle 1of hepatocyte growth factor; rhuHGF recombinant human hepatocyte growth factor; PAGE, polyacrylamide gel Escherichia coli.
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