Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains.
Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.
Okigaki
M
M
Institute for Liver Research, Kansai Medical University, Osaka, Japan.
Komada
M
M
Uehara
Y
Y
Miyazawa
K
K
Kitamura
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D017228:Hepatocyte Growth Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met; D011956:Receptors, Cell Surface / Q000378:metabolism; D017384:Sequence Deletion; D014443:Tyrosine / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D014443:Tyrosine; D017228:Hepatocyte Growth Factor; D004247:DNA; D019859:Proto-Oncogene Proteins c-met