FUNCTIONAL-CHARACTERIZATION OF HUMAN HEPATOCYTE GROWTH-FACTOR MUTANTS OBTAINED BY DELETION OF STRUCTURAL DOMAINS
FUNCTIONAL-CHARACTERIZATION OF HUMAN HEPATOCYTE GROWTH-FACTOR MUTANTS OBTAINED BY DELETION OF STRUCTURAL DOMAINS
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DOI：
10.1021/bi00155a007
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发表时间：
1992-10-13
期刊：
影响因子：
2.9
通讯作者：
KITAMURA, N
中科院分区：
文献类型：
--
作者：
OKIGAKI, M;KOMADA, M;KITAMURA, N
人肝细胞生长因子(HHGF)由独特的结构域组成。在这项研究中，我们制备了缺乏这些结构域的突变蛋白，并检测了它们刺激肝细胞DNA合成、抑制Meth A细胞生长和诱导MDCK细胞解离的生物学活性。我们还通过竞争分析和酪氨酸磷酸化水平分析检测了它们与c-met/HGF受体的相互作用。缺失N端、第一Kringle结构域或第二Kringle结构域的突变蛋白不具有生物学活性，不能与c-Met/HGF受体结合的hHGF竞争。结果表明，这些结构域是hHGF与c-Met/HGF受体结合所介导的生物活性所必需的。缺乏第三或第四kringle结构域的突变蛋白适度地保留了生物活性和受体结合。与野生型hHGF相比，这些突变蛋白对c-Met/HGF受体酪氨酸磷酸化的相对水平与生物活性的相对强弱有很好的相关性。缺失轻链的突变蛋白对c-Met/HGF受体的生物活性和酪氨酸磷酸化没有影响，但与c-Met/HGF受体结合的hHGF竞争。这些结果表明，重链在hHGF与c-Met/HGF受体的相互作用中起着重要作用，而轻链则是c-Met/HGF受体酪氨酸磷酸化所必需的。
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.