Ermanno Gherardi

Met, metastasis, motility and more

Hepatocyte growth factor/scatter factor and its receptor, the tyrosine kinase Met, arose late in evolution and are unique to vertebrates. In spite of this, Met uses molecules such as Gab1 — homologues of which are present in Caenorhabditis elegans and Drosophila melanogaster — for downstream signalling. Pivotal roles for Met in development and cancer have been established: Met controls cell migration and growth in embryogenesis; it also controls growth, invasion and metastasis in cancer cells; and activating Met mutations predispose to human cancer.

Targeting MET in cancer: rationale and progress

Uncontrolled cell survival, growth, angiogenesis and metastasis are essential hallmarks of cancer. Genetic and biochemical data have demonstrated that the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, have a causal role in all of these processes, thus providing a strong rationale for targeting these molecules in cancer. Parallel progress in understanding the structure and function of HGF/SF, MET and associated signalling components has led to the successful development of blocking antibodies and a large number of small-molecule MET kinase inhibitors. In this Review, we discuss these advances, as well as results from recent clinical studies that demonstrate that inhibiting MET signalling in several types of solid human tumours has major therapeutic value.

Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase

A number of developmental processes that involve cell migration, growth or morphogenesis depend on extracellular signals. A molecule that provides such signals, known as hepatocyte growth factor/scatter factor (HGF/SF), has attracted considerable interest in recent years because of its distinct structure, mechanism of activation and important roles throughout embryogenesis. This review discusses the main features of HGF/SF and its receptor, the product of the c-met protooncogene, and their role in embryogenesis.

Structural repertoire of the human VH segments.

The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individuals gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.

The structural repertoire of the human V kappa domain.

In humans, the gene for the V kappa domain is produced by the recombination of one of 40 functional V kappa segments and one of five functional J kappa segments. We have analysed the sequences of these germline segments and of 736 rearranged V kappa genes to determine the repertoire of main chain conformations, or canonical structures, they encode. Over 96% of the sequences correspond to one of four canonical structures for the first antigen binding loop (L1) and one canonical structure for the second antigen binding loop (L2). Junctional diversity produces some variation in the length of the third antigen binding loop (L3) and in the identity of residues at the V kappa‐J kappa join. However, this is limited and 70% of the rearranged sequences correspond to one of three known canonical structures for the L3 region. Furthermore, we show that the canonical structures selected during the primary response are conserved during affinity maturation: the key residues that determine the conformations of the antigen binding loops are unmutated or undergo conservative mutation. The implications of these results for immune recognition are discussed.

Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor

Little is known about the large ectodomain of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor (HGF/SF). Here, we establish by deletion mutagenesis that the HGF/SF and heparin-binding sites of MET are contained within a large N-terminal domain spanning the α-chain (amino acids 25–307) and the first 212 amino acids of the β-chain (amino acids 308–519). Neither the cystine-rich domain (amino acids 520–561) nor the C-terminal half of MET (amino acids 562–932) bind HGF/SF or heparin directly. The MET ectodomain, which behaves as a rod-shaped monomer with a large Stokes radius in solution, binds HGF/SF in the absence or presence of heparin, and forms a stable HGF/SF–heparin–MET complex with 1:1:1 stoichiometry. We also show that the ligand-binding domain adopts a β-propeller fold, which is similar to the N-terminal domain of αV integrin, and that the C-terminal half contains four Ig domains (amino acids 563–654, 657–738, 742–836, and 839–924) of the unusual structural E set, which could be modeled on bacterial enzymes. Our studies provide 3D models and a functional map of the MET ectodomain. They have broad implications for structure-function of the MET receptor and the related semaphorin and plexin proteins.

Structure of the Human Receptor Tyrosine Kinase Met in Complex with the Listeria Invasion Protein InlB

The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.

Diverse and potent activities of HGF/SF in skin wound repair†

Genetic studies in the mouse have highlighted essential roles for several growth factors in skin repair and have offered a rationale for their use in therapy. The present study shows that the plasminogen‐related growth factor HGF/SF (hepatocyte growth factor/scatter factor) promotes wound repair in homozygous diabetic db/db mice by recruiting neutrophils, monocytes, and mast cells to the wound; by promoting the migration of endothelial cells to the injured area; and by enhancing keratinocyte migration and proliferation. As a result, granulation tissue formation, wound angiogenesis, and re‐epithelialization are all increased. The results demonstrate that HGF/SF affects and sustains all key cellular processes responsible for wound repair and point to a unique potential of this molecule for the therapy of chronic skin wounds. Copyright

Hepatocyte Growth Factor/Scatter Factor Can Induce Angiogenesis Independently of Vascular Endothelial Growth Factor

Objective—Hepatocyte growth factor/scatter factor (HGF/SF) promotes vascular endothelial growth factor (VEGF) expression and induces angiogenesis in multiple pathological conditions. The present study was designed to delineate the HGF/SF and VEGF signaling cascades during angiogenesis by using PTK787, a selective VEGF receptor antagonist. Methods and Results—PTK787 produced a concentration-dependent (10−8 to 10−6 mol/L) inhibition of VEGF-induced angiogenesis, without altering the basal or HGF/SF-induced response in vitro. In contrast, the nonspecific kinase inhibitor genistein blocked the HGF/SF-induced effect. Both VEGF and HGF/SF induced a rapid phosphorylation of extracellular receptor kinases-1 and -2 (ERKs) and Akt. PTK787 inhibited the VEGF-induced activation of Akt and ERKs, without affecting the HGF/SF-induced phosphorylation. Treatment with VEGF and HGF/SF increased total neovascularization in a murine scaffold granuloma model, but no additive or synergistic interactions were observed. PTK787 (50 mg/kg) blocked the VEGF-induced response without altering the basal or HGF/SF-induced neovascularization. Conclusions—We demonstrate that HGF/SF can induce angiogenesis independently of VEGF, possibly through the direct activation of the Akt and ERKs. These results demonstrate the necessity of a multitargeted approach for the rational design of newer therapies to inhibit pathophysiological angiogenesis.

Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding

Although ligand-induced receptor dimerization is a common prerequisite for receptor activation, the mode by which different growth factors bind their receptors and cause them to dimerize varies considerably. Here we report the crystal structure at 2.5 Å resolution of NK1, a receptor-binding fragment and a natural splice variant of hepatocyte growth factor/scatter factor (HGF/SF). NK1 assembles as a homodimer in the asymmetric unit, revealing a novel mode of growth factor dimerization produced by close packing of the N domain of one subunit and the kringle domain of the other, thus bringing the two linkers in close proximity. The structure suggests the presence of a binding site for heparan sulfate chains and a mechanism by which the NK1 dimer may engage two receptor molecules through clusters of amino acids located on each protomer and on opposite surfaces of the homodimer. We also report that short (14-mer) heparin fragments effectively dimerize NK1 in solution, implying that heparan sulfate chains may stabilize the NK1 dimer. These results provide a basis for the agonistic activity of NK1 and have implications for the mechanism of receptor binding of HGF/SF.