Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.
Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.
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原代培养的成年大鼠肝细胞上两类人肝细胞生长因子受体的鉴定和部分表征。
DOI：
10.1016/s0021-9258(19)50543-9
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Y. Daikuhara
中科院分区：
文献类型：
--
作者：
N. Arakaki;Shuichi Hirono;T. Ishii;Masayoshi Kimoto;S. Kawakami;H. Nakayama;H. Tsubouchi;T. Hishida;Y. Daikuhara
人肝细胞生长因子(HHGF)是一种参与肝损伤后肝再生的生理性肝营养因子，为了研究其受体(S)的特性，将重组人肝细胞生长因子(HHGF)进行放射性标记。在原代培养中，标记的重组人肝细胞生长因子在成年大鼠肝细胞上保持了其全部生物学活性。[125I]IODO-rhHGF与肝细胞的结合在4℃的240分钟内达到平台期。结合数据的Scatchard图分析表明，存在两类高亲和力结合位点。其中一个的解离常数(Kd)约为4.6 pm，约为300个位点/cell，另一个的解离常数(Kd)约为275 pm，约为15,160个位点/cell。未标记的重组人肝细胞生长因子以剂量依赖的方式取代细胞表面结合的[125I]碘-重组人肝细胞生长因子，从暴发性肝衰竭患者的血浆中纯化的天然人肝细胞生长因子也是如此。然而，原代培养的大鼠肝细胞的其他生长因子如胰岛素和人表皮生长因子，以及与hHGF氨基酸序列同源性较高的蛋白质如纤溶酶原和凝血酶原等，并不与[125I]碘-rhHGF竞争结合，这表明这种结合是hHGF特异性的。[~(125)I]碘-重组人肝细胞生长因子与肝细胞表面受体(S)的共价交联实验表明，肝细胞表面有两个大分子物种，表观分子量分别为330,000和230,000。未标记的重组人肝细胞生长因子和天然人肝细胞生长因子竞争结合[125I]碘-重组人肝细胞生长因子，而胰岛素、人表皮生长因子、纤溶酶原和凝血酶原不结合。根据我们估计的重组人肝细胞生长因子的相对分子质量为84,000，这些结果表明，人肝细胞生长因子与两个分别代表原代培养大鼠肝细胞人肝细胞生长因子受体的多肽(246,000和146,000道尔顿)特异性结合。
To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.