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.
Lokker
N A
NA
Department of Cell Genetics, Genentech, Inc., South San Francisco, California 94080.
Godowski
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D017228:Hepatocyte Growth Factor / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D004247:DNA; D019859:Proto-Oncogene Proteins c-met