Functional analysis of domain structures of human hepatocyte growth factor

人肝细胞生长因子（hHGF）是一种多功能蛋白质。hHGF由特征性结构域组成;其在重链中具有四个kringle结构域，在轻链中具有丝氨酸蛋白酶样结构域。为了阐明这些结构域结构的作用，我们制备了缺乏这些结构域的突变蛋白，并研究了它们刺激肝细胞DNA合成、抑制MethA细胞生长和诱导MDCK细胞解离的生物活性。我们还研究了它们与c-met/HGF受体的相互作用，通过置换分析和酪氨酸磷酸化水平的分析。缺失N末端、第一Kringle结构域或第二Kringle结构域的突变蛋白没有生物学效果，并且不能置换与c-met/HGF受体结合的hHGF。结果表明，这些结构域是必要的hHGF的生物活性介导的结合c-met/HGF受体。缺失第三或第四Kringle结构域的突变蛋白适度保留生物活性和受体结合。与野生型hHGF相比，这些突变体蛋白对c-met/HGF受体酪氨酸磷酸化的相对水平与生物活性的相对效力相关。缺失轻链的突变蛋白对c-met/HGF受体的生物学活性和酪氨酸磷酸化没有影响，但取代了与c-met/HGF受体结合的hHGF。这些结果表明重链在hHGF与c-met/HGF受体的相互作用中起重要作用，并且轻链对于c-met/HGF受体的酪氨酸磷酸化是进一步需要的。

Human hepatocyte growth factor (hHGF) is a multi-functional protein. hHGF consists of characteristic structural domains ; it has four kringle domains in the heavy chain and a serine proteaselike domain in the light chain. To elucidate the role of these domain structures, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of MethA cell growth and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by displacement analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the Nterminal, the first kringle or the second kringle domain were not biologically effective and could not displace 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 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 displaced 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.