IDENTIFICATION AND PARTIAL CHARACTERIZATION OF 2 CLASSES OF RECEPTORSFOR HUMAN HEPATOCYTE GROWTH-FACTOR ON ADULT-RAT HEPATOCYTES IN PRIMARY CULTURE

To characterize the receptor(s) for human hepatocyte growth factor (hH GF), a physiological hepatotrophic factor involved in liver regenerati on following hepatic injury, recombinant hHGF (rhHGF) was radioiodinat ed. The labeled rhHGF retained its full biological activity on adult r at hepatocytes in primary culture. The specific binding of [I-125]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 [I-125]iodo-rhHGF. One of the sites had a dissociation constant (K(d)) of about 4.6 pM with 300 sites/cell and the other has a K(d) of about 275 pM with 15,160 si tes/cell. Unlabeled rhHGF displaced cell surface-bound [I-125]iodo-rhH GF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth fact ors to rat hepatocytes in primary culture such as insulin and human ep idermal growth factor, and proteins which have high amino acid sequenc e-homology to hHGF such as plasminogen and prothrombin, did not compet e with [I-125]iodo-rhHGF in the binding, which suggests the binding wa s specific to hHGF. Covalent cross-linking experiment of [I-125]iodo-r hHGF 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 [ I-125]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 s uggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of p rimary cultured rat hepatocytes.