IDENTIFICATION OF FUNCTIONAL DOMAINS IN THE HEPATOCYTE GROWTH-FACTOR AND ITS RECEPTOR BY MOLECULAR ENGINEERING

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide which shares structural domains with enzymes of the blood clotting cascade. HGF/SF is secreted by cells of mesodermal origin and has powerful mitogenic, motogenic and morphogenic activity on epithel ial and endothelial cells. HGF/SF is produced as a biologically inacti ve single-chain precursor (pro-HGF/SF) most of which is sequestered on the cell surface or bound to the extracellular matrix. Maturation int o the active alpha beta heterodimer results from proteolytic cleavage by a urokinase-type protease, which acts as a pro-HGF/SF convertase. T he primary determinant for receptor binding appears to be located with in the alpha-chain. The interaction of the alpha-chain with the recept or is sufficient for the activation of the signal cascade involved in the motility response. However, the complete HGF/SF protein seems to b e required to elicit a mitogenic response. HGF/SF binds with high affi nity to a transmembrane receptor, p190(MET), encoded by the MET proto- oncogene. p190(MET) is the prototype of a distinct subfamily of hetero dimeric tyrosine kinases, including the putative receptors Ron and Sea , The mature form of p190(MET) is a heterodimer of two disulfide-linke d subunits (alpha and beta). The alpha-subunit is extracellular and he avily glycosylated. The beta-subunit consists of an extracellular port ion involved in ligand binding, a membrane spanning segment, and a cyt oplasmic tyrosine kinase domain. Both subunits derive from glycosylati on and proteolytic cleavage of a common precursor of 170 kDa. In polar ized epithelial cells the HGF/SF receptor is selectively exposed in th e basolateral plasmalemma, where it is associated with detergent-insol uble components. Two Met isoforms, carrying an intact ligand binding d omain but lacking the kinase domain due to truncation of the beta-subu nit, arise from alternative post-transcriptional processing of the mat ure form. One truncated form is soluble and released from the cells. H GF/SF binding triggers tyrosine autophosphorylation of the receptor be ta-subunit. Autophosphorylation on the major phosphorylation site Y-12 35 upregulates the kinase activity of the receptor, increasing the V-m ax of the phosphotransfer reaction. Negative regulation of the kinase activity occurs through phosphorylation of a unique serine residue (S- 985) located in the juxtamembrane domain of the receptor. This phospho rylation is triggered by two distinct pathways involving either protei n kinase C activation or increase in intracellular Ca2+ concentration.