Generation and characterization of a competitive antagonist of human hepatocyte growth factor, HGF/NK1.
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.[1]References
- Generation and characterization of a competitive antagonist of human hepatocyte growth factor, HGF/NK1. Lokker, N.A., Godowski, P.J. J. Biol. Chem. (1993) [Pubmed]
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