Molecular oncology. Causes of cancer and targets for treatment. Eds Edward P. Gelmann et al., Cambridge University Press (2014)
The human HGF gene consists of 18 exons and 16 introns spanning 68 Mb on chromosome 7q21.11 (1). Five mRNA transcripts arise from alternative splicing: two encode fulllength HGF forms and three encode truncated isoforms that bind Met, but differ in their biological activities (1). HGF protein is a plasminogen family member consisting of an aminoterminal heparin-binding domain (N), four kringle domains (K1–4) and a carboxyl-terminal serine-protease-like domain (Figure 17.1a). Unlike other plasminogen family members, HGF has no proteolytic activity (1). The HGF N and K1 domains contain the primary Met binding sites (12), and the protease-like domain contains an important secondary Met binding site (13). Proteolytic processing of the singlechain HGF precursor results in the active disulfide-linked heterodimer; the amino-terminal -chain contains N and K1–4, and the -chain contains the protease-like region (1).
The human MET oncogene was isolated from a human osteosarcoma-derived cell line on the basis of its transforming activity in vitro, caused by a DNA rearrangement where sequences from the TPR (translocated promoter region) locus on chromosome 1 were fused to part of the MET sequence on chromosome 7 (TPR-MET; 2). A similar rearrangement was later found in patients with gastric carcinoma (2,14). The full-length MET gene on chromosome 7q21–q31 comprises 21 exons separated by 20 introns spanning 120 kb, and encodes the receptor known as Met (or cellular-Met, c-Met; 15). The primary transcript produces a 150 kDa polypeptide that is glycosylated to produce a 170 kDa precursor, then further glycosylated to 190 kDa before cleavage into a disulfide-linked 50 kDa -chain and a 140 kDa -chain (Figure 17.1b; 5). The entire -chain and amino-terminal portion of the -chain forms a semaphorin (Sema) domain with a seven-bladed –propeller fold (13,16), also found in semaphorin receptors (17,18). This domain, as well as the more highly conserved TK domain, places Met in a subfamily with Ron (also known as Sea; 16).
Carboxyl-terminal to the Sema domain is the PSI domain (found in plexins, semaphorins, and integrins), and then four immunoglobulin (Ig)-like domains (16). The Sema domain is the primary HGF-binding domain in Met (16), although Ig-domains 3 and 4, located closest to the transmembrane domain, reportedly also mediate high-affinity HGF binding (19). Like all receptor TKs, the Met transmembrane domain is thought to consist of a single -helix (20). The most aminoterminal cytoplasmic subdomain, the juxtamembrane (JM) region, contains two phosphorylation sites: S985 and Y1003 (all numbering per SwissProt Accession P08581). Phosphorylation of S985 negatively regulates kinase activity, whereas phosphorylation of Y1003 recruits c-Cbl, which monoubiquinates Met and interacts with endophilin, targeting Met for internalization and degradation (2). The protein tyrosine phosphatase PTP-S also binds to this region (21). Carboxyl-terminal to the JM region is the TK domain, which shares homology with insulin growth factor I receptors and the Tyro 3 family of immunoregulatory molecules, and lastly, a carboxylterminal tail region. Upon HGF binding, Met autophosphorylation occurs on tyrosines Y1234 and Y1235 within the TK domain activation loop, enhancing kinase activity, and on Y1349 and Y1356 in the carboxyl-terminal region, which forms a multi-functional docking site for a collection of adaptor proteins containing Src homology-2 (SH2) domains and other conserved recognition motifs (22). An intact multi-functional docking site is required to mediate transformation and induce a metastatic phenotype (23).
The key adaptors and direct kinase substrates thus far implicated in Met signaling are Grb2, Gab1, phosphatidylinositol 3-kinase (PI3K), phospholipase C (PLC ), Shc, Src, Shp2, Ship1, and STAT3 (Figure 17.1c; 2,5). Among these, Gab1, Grb2, PI3K, Shc, and Src can bind Met directly; through them, a larger network of adaptor proteins are involved in signaling. In particular, Grb2 binding at Met Y1356 links Met to the Ras/MAPK pathway regulating cell-cycle progression (5). Gab1 is recruited to Met through direct binding and indirectly via Grb2; these interactions initiate branching morphogenesis in several epithelial and vascular endothelial cell types (24). Gab1 is also highly phosphorylated by Met, resulting in the additional recruitment of PI3K, contributing in turn to cell survival, cell-cycle progression, and motility (2–6).