Cancer Therapy Vol 3, page 3 a unique AL conformation. This conformation may represent a quasi-stable intermediate state along the transition pathway between phosphorylated and unphosphorylated AL conformations, although it is possible this conformation is an artifact arising from mutations to the conserved residues within the AL.
the binding stoichiometry of EGF to receptor was 1:1, and the dimeric complex was stabilized solely through receptor-receptor interactions formed upon ligand binding (Ogiso et al, 2002). The mechanism utilized by HGF to induce c-MET dimerization remained largely elusive until recently, when the crystal structure of the HGF "-chain and c-MET sema domain highlighted a possible dimer interface between the ligand-receptor pair, and suggested a potential 2:2 HGF:c-MET complex (Stamos et al, 2004). Future structural studies on intact HGF and c-MET ectodomain would shed light on the structural-basis of recruitment of HGF by c-MET, and subsequent c-MET dimerization induced by HGF. Interestingly, cross-linking c-MET receptors by specific antibodies to the extracellular domain can trigger c-MET signaling implying that dimerization is sufficient to activate c-MET (Prat et al, 1998). Irrespective of its mode of dimerization, autophosphorylation of c-MET tyrosines necessary for signaling occurs after dimerization and presumably, by transphosphorylation between the catalytic domains of dimeric c-MET. The biochemical events regulating c-MET signaling have been recently elucidated (as discussed below), although, the structural basis for c-MET autophosphorylation upon HGF binding remains largely unclear. Tyrosines Y1231, Y1234 and Y1235 in the AL of the c-MET catalytic domain have been shown to be phosphorylated in response to HGF-induced c-MET dimerization (Figure 1). The presence of three phosphotyrosine sites in the AL is also a characteristic of the insulin receptor, a disulfide-linked constitutive dimer. While the phosphorylation of AL tyrosines is important for increased c-MET kinase activity (Rodrigues et al, 1994), the phosphorylation of carboxy-terminal tail tyrosines Y1349, Y1356 and Y1365 (Birchmeier et al, 2003) is required for the recruitment of cytoplasmic signaling proteins with Src homology-2 (SH2) and protein tyrosine binding (PTP) domains. Phenylalanine substitution at residues Y1349 and Y1356 render c-MET functionally impaired in its ability to induce proliferation, motility, differentiation and survival (Weidner et al, 1995). In addition, phosphorylation of Y1003 within the juxtamembrane region appears to be critical for receptor degradation (Peschard et al, 2001, 2004).
B. HGF/SF structure The ligand for c-MET was independently identified by two different laboratories as a mitogen for hepatocytes, HGF and a SF in fibroblasts (Stoker et al, 1986, 1987; Nakamuraet al, 1989). Since its discovery, HGF has been shown to elicit plietropic cellular responses including mitogenesis, motility and morphogenesis. HGF/SF is synthesized as an inactive single-chain precursor that is proteolytically cleaved to form an active disulfide-linked heterodimer. Both the single-chain precursor as well as disulfide-linked heterodimer appear to bind c-MET with high affinity, however, c-MET activation occurs only by the cleaved mature form of the ligand (Lokker et al, 1992). HGF shows sequence homology to the plasminogenrelated growth factor family: these proteins have a similar cleavage-mediated activation mechanism. The 69 kDa !chain of HGF consists of an N-terminal domain (N), followed by four kringle domains (K1-K4) (Lokker et al, 1992). The 34 kDa "-chain forms a conserved proteaselike domain; this domain is inactive due to substitution of required active site serine and histidine residues. The HGF residues that form the receptor binding site are unknown, although a number of studies indicate that the !- and "chains have distinct roles in c-MET binding, and subsequent dimerization (Ultsch et al, 1998; Lietha et al, 2001; Gherardi et al, 2003; Stamos et al, 2004). Several truncated forms of the !-chain region, including NK1, bind c-MET with high affinity. HGF is also a high-affinity ligand for heparan sulfate proteoglycans. However, unlike fibroblast growth factor receptor (FGFR), this interaction does not appear to be critical for c-MET activation (Lokker et al, 1992; DiGabriele et al, 1998; Hartmannet al, 1998). The "-chain of HGF, which harbors the serineprotease-like catalytic domain, has also been shown to bind the sema domain within c-MET, albeit with relatively lower affinity (Stamos et al, 2004).
C. HGF-induced c-MET dimerization and activation
III. c-MET functions A. c-MET signaling
Normal signaling by RTKs requires ligand-induced receptor oligomerization and tyrosine phosphorylation of the cytoplasmic domains of the receptor. Although ligandmediated receptor dimerization appears to be a common event preceding RTK activation, structures of several receptor ectodomains bound to their cognate ligands showed RTKs used different binding modes to accomplish dimerization. In vascular endothelial growth factor receptor (VEGFR), dimerization was induced by binding of dimeric ligand (Wiesmann et al, 1997), whereas FGFR bound monomeric FGF and the dimeric complex was stabilized by heparin cofactors (Schlessinger et al, 2000; Mohammadi et al, 2005). Recent crystallographic studies of epidermal growth factor receptor (EGFR) showed that
Functional genetic studies of c-MET and HGF have conclusively revealed an indispensable role of these molecules in mammalian development. HGF -/- and cMET -/- mice die in utero after incurring severe placental and live defects, along with disruption in the migration of myogenic precursors into the limb bud (Bladt et al, 1995; Schmidt et al, 1995; Uehara et al, 1995). Furthermore, in adults, c-MET and HGF are widely expressed, and c-MET signaling has been shown to be important for tissue repair and organ regeneration (Michalopoulos et al, 1997; Matsumoto et al, 2001). In the recent years, extensive studies have been conducted to elucidate the mechanism by which HGF/c-MET regulate such diverse physiological responses. 3