Fold and function of the InlB B-repeat

Host cell invasion by the facultative intracellular pathogen Listeria monocytogenes requires the invasion protein InlB in many cell types. InlB consists of an N-terminal internalin domain that binds the host cell receptor tyrosine kinase Met and C-terminal GW domains that bind to glycosaminoglycans (GAGs). Met binding and activation is required for host cell invasion, while the interaction between GW domains and GAGs enhances this effect. Soluble InlB elicits the same cellular phenotypes as the natural Met ligand hepatocyte growth factor/scatter factor (HGF/SF), e.g. cell scatter. So far, little is known about the central part of InlB, the B-repeat. Here we present a structural and functional characterization of the InlB B-repeat. The crystal structure reveals a variation of the β-grasp fold that is most similar to small ubiquitin-like modifiers (SUMOs). However, structural similarity also suggests a potential evolutionary relation to bacterial mucin-binding proteins. The B-repeat defines the prototype structure of a hitherto uncharacterized domain present in over a thousand bacterial proteins. Generally, this domain probably acts as a spacer or a receptor-binding domain in extracellular multi-domain proteins. In cellular assays the B-repeat acts synergistically with the internalin domain conferring to it the ability to stimulate cell motility. Thus, the B-repeat probably binds a further host cell receptor and thereby enhances signaling downstream of Met.     

Structural insights into Met receptor activation

The receptor tyrosine kinase Met plays a pivotal role in vertebrate development and tissue regeneration, its deregulation contributes to cancer. Met is also targeted during the infection by the facultative intracellular bacterium Listeria monocytogenes. The mechanistic basis for Met activation by its natural ligand hepatocyte growth factor/scatter factor (HGF/SF) is only beginning to be understood at a structural level. Crystal structures of Met in complex with L. monocytogenes InlB suggest that Met dimerization by this bacterial invasion protein is mediated by a dimer contact of the ligand. Here, I review the structural basis of Met activation by InlB and highlight parallels and differences to the physiological Met ligand HGF/SF and its splice variant NK1.     

Ligand-Mediated Dimerization of the Met Receptor Tyrosine Kinase by the Bacterial Invasion Protein InlB

The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.     

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.     

InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase

The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.     

Structural basis of MET receptor dimerization by the bacterial invasion protein InlB and the HGF/SF splice variant NK1

The structural basis of ligand-induced dimerization of the receptor tyrosine kinase MET by its natural ligand hepatocyte growth factor/scatter factor (HGF/SF) is not well understood. However, interesting insight into the molecular mechanism of MET dimerization has emerged from crystal structures of MET in complex with a bacterial agonist, the invasion protein internalin B (InlB) from pathogenic Listeria monocytogenes. MET activation by InlB promotes uptake of bacteria into host cells. Structural and biophysical data suggest that InlB is monomeric on its own but dimerizes upon binding to the membrane-anchored MET receptor promoting the formation of a signaling active 2:2 complex. The dimerization interface is small and unusually located on the convex side of the curved InlB leucine-rich repeat (LRR) domain. As InlB does not dimerize in solution, the dimerization site could only be identified by studying packing contacts of InlB in various crystal forms and had to be proven by scrutinizing its biological relevance in cellular assays. InlB dimerization is thus an example of a low-affinity contact that appears irrelevant in solution but becomes physiologically significant in the context of 2-dimensional diffusion restricted to the membrane plane. The resulting 2:2 InlB:MET complex has an InlB dimer at its center with one MET molecule bound peripherally to each InlB. This model of ligand-mediated MET dimerization may serve as a blue-print to understand MET activation by NK1, a naturally occurring HGF/SF splice variant and MET agonist. Crystal structures of NK1 repeatedly show a NK1 dimer, in which residues implicated in MET-binding are located on the outside. Thus, MET dimerization by NK1 may also be ligand-mediated with a NK1 dimer at the center of the 2:2 complex with one MET molecule bound peripherally to each NK1. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation

Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB??????) was demonstrated to bind to and partially activate the HGF receptor Met. InlB?????? has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB?????? (1×InlB??????) and engineered a head-to-tail repeat of InlB?????? with a linker peptide (2×InlB??????); 1×InlB?????? and 2×InlB?????? were purified from E. coli. Both 1× and 2×InlB?????? activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB?????? activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB?????? activated only STAT3 and ERK1/2. The 2×InlB?????? promoted improved motility compared with 1×InlB?????? and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB?????? prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB?????? to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.     

Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains

Hepatocyte growth factor/scatter factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the beta-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (K(d)=2.4, 3.3 and 1.4 microM). The same domains also bound heparin with decreasing affinities (N>K1>SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the beta-chain moiety (amino acid residues 308-514) of the Met beta-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy.     

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB₃₂₁) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB₃₂₁ consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB₃₂₁ in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB₃₂₁ binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB₃₂₁. These results call into question whether receptor dimerization is the basic underlying event in InlB₃₂₁-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB₃₂₁ bind and activate the Met receptor.     

Synergy between the N- and C-terminal domains of InlB for efficient invasion of non-phagocytic cells by Listeria monocytogenes

InlB is a Listeria monocytogenes protein promoting entry in non-phagocytic cells, and has been shown recently to activate the hepatocyte growth factor receptor (HGFR or Met). The N-terminal domain of InlB (LRRs) binds and activates Met, whereas the C-terminal domain of InlB (GW modules) mediates loose attachment of InlB to the listerial surface. As HGF activation of Met is tightly controlled by glycosaminoglycans (GAGs), we tested if GAGs also modulate the Met-InlB interactions. We show that InlB-dependent invasion of non-phagocytic cells decreases up to 10 times in the absence of GAGs, and that soluble heparin releases InlB from the bacterial surface and promotes its clustering. Furthermore, we demonstrate that InlB binds cellular GAGs by its GW modules, and that this interaction is required for efficient InlB-mediated invasion. Therefore, GW modules have an unsuspected dual function: they attach InlB to the bacterial surface and enhance entry triggered by the LRRs domain. Our results thus provide the first evidence for a synergy between two host factor-binding domains of a bacterial invasion protein, and reinforce similarities between InlB and mammalian growth factors.     

Involvement of hepatocyte growth factor/scatter factor and met receptor signaling in hair follicle morphogenesis and cycling.

HGF/SF and its receptor (Met) are principal mediators of mesenchymal-epithelial interactions in several different systems and have recently been implicated in the control of hair follicle (HF) growth. We have studied their expression patterns during HF morphogenesis and cycling in C57BL/6 mice, whereas functional hair growth effects of HGF/SF were assessed in vivo by analysis of transgenic mice and in skin organ culture. In normal mouse skin, follicular expression of HGF/SF and Met was strikingly localized: HGF/SF was found only in the HF mesenchyme (dermal papilla fibroblasts) and Met in the neighboring hair bulb keratinocytes. Both HGF/SF and Met expression peaked during the initial phases of HF morphogenesis, the stage of active hair growth (early and mid anagen), and during the apoptosis-driven HF regression (catagen). Met+ cells in the regressing epithelial strand appeared to be protected from undergoing apoptosis. Compared to wild-type controls, transgenic mice overexpressing HGF/SF under the control of the MT-1 promoter had twice as many developing HF and displayed accelerated HF development on postnatal day 3. They also showed significant catagen retardation on P17. In organ culture and in vivo, HGF/SF i.c. resulted in a significant catagen retardation. These results demonstrate an important role of HGF/SF and Met in murine hair growth control and suggest that Met-mediated signaling might be exploited for therapeutic manipulation of human hair growth disorders.-Lindner, G., Menrad, A., Gherardi, E., Merlino, G., Welker, P., Handjiski, B., Roloff, B., Paus, R. Involvement of hepatocyte growth factor/scatter factor and Met receptor signaling in hair follicle morphogenesis and cycling.     

Radioimmunoscintigraphy of tumors autocrine for human met and hepatocyte growth factor/scatter factor

Inappropriate expression of the c-met-protooncogene product (Met) and/or of its ligand, hepatocyte growth factor/scatter factor (HGF/SF), has been correlated with poor prognosis in a variety of human solid tumors. We are developing animal models for nuclear imaging of Met and HGF/SF expression in tumors in vivo. We radioiodinated a mixture of monoclonal antibodies (MAbs) that bind to human HGF/SF and to the external ligand-binding domain of human Met, and then injected the I-125-MAb mixture intravenously into mice bearing tumors either autocrine for human HGF/SF and human Met or autocrine-paracrine for murine HGF/SF and murine Met. Serial total body gamma camera images were obtained, and regional activity was determined by quantitative region-of-interest (ROI) analysis. Tumors autocrine for human HGF/SF and Met demonstrated significantly more rapid uptake and more rapid clearance of the I-125-MAb mixture than tumors expressing one or both murine homologues, reaching a mean tumor to total body activity ratio of > 0.3 by 1 day postinjection. We conclude that radioimmunodetection of tumors autocrine for human HGF/SF and Met is feasible with an I-125-MAb mixture reactive against the ligand-receptor pair.     

The Listeria protein internalin B mimics hepatocyte growth factor-induced receptor trafficking

Increased hepatocyte growth factor receptor (HGFR) signaling correlates closely with neoplastic invasion and metastatic potential of many human cancers. Hepatocyte growth factor receptor signaling is initiated by binding the physiological ligand HGF or the internalin B (InlB) protein of Listeria monocytogenes. Subsequent degradation of endocytosed HGFR terminates receptor signaling. Previously reported discrepancies in InlB and HGF-induced HGFR signaling could reflect differences in receptor internalization and degradation in response to these distinct ligands. We report that soluble InlB and HGF are mechanistically equivalent in triggering clathrin-dependent endocytosis and lysosomal degradation of HGFR. After internalization, InlB and HGF colocalize with Rab5, EEA1 and the transferrin receptor in classical early endosomes. Hepatocyte growth factor receptor internalization was prevented by overexpression of dominant negative mutants of dynamin 1 and epidermal growth factor phosphorylation substrate 15, but not caveolin 1, the GTPase Arf6 or the cholesterol-chelating drug Nystatin. Thus, HGFR internalization is principally clathrin-mediated and is not regulated by clathrin- independent pathways. Phosphatidylinositol 3-kinase signaling and HGF-regulated tyrosine kinase substrate were not required for ligand-triggered internalization of HGFR but were essential for subsequent lysosomal degradation. Thus, soluble InlB and HGF induce HGFR endocytosis and degradation by indistinguishable mechanisms, suggesting that InlB may be exploited to regulate pathogenic HGFR signaling.     

Crystal structure of the HGF beta-chain in complex with the Sema domain of the Met receptor

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF-dependent signaling requires proteolysis from an inactive single-chain precursor into an active alpha/beta-heterodimer. We show that the serine protease-like HGF beta-chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven-bladed beta-propeller, where the bottom face of blades 2 and 3 binds to the HGF beta-chain 'active site region'. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF beta binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF beta-chains brings two HGF beta:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.     

Multiple regions of internalin B contribute to its ability to turn on the Ras-mitogen-activated protein kinase pathway

Internalin B (InlB) is a protein present on the surface of Listeria monocytogenes that mediates bacterial entry into mammalian cells. It is thought that InlB acts by binding directly to the hepatocyte growth factor (HGF) receptor, present on the surface of host cells. Binding of InlB to the HGF receptor results in mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase activation, followed by changes in the organization of the actin cytoskeleton. Here we have compared signaling by HGF and InlB. Whereas stimulation with equivalent concentrations of HGF and InlB elicits similar activation of the HGF receptor, we observed striking differences in downstream activation of MAP kinase. InlB leads to a greater activation of the Ras-MAP kinase pathway than does HGF. The leucine-rich repeat region, which was previously shown to be sufficient for binding and activation of the HGF receptor, lacks the ability to super-activate the Ras-MAP kinase pathway. Analysis of a series of deletion mutants suggests that it is the B repeat region between the leucine-rich repeat and GW domains that endows InlB with an increased ability to turn on the Ras-MAP kinase pathway. These unexpected observations suggest that HGF and InlB use alternative mechanisms to turn on cellular signaling pathways.     

GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation

The Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met. Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond. In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N-terminal LRR region and C-terminal GW domains. The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans. Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor-mediated mode, as opposed to the ligand-mediated mode observed for the LRR fragment. Surprisingly, we find that gC1q-R, a host protein implicated in InlB-mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q-R is unnecessary for bacterial invasion. Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone-like mimicry of HGF.     

HGF/SF-met signaling in the control of branching morphogenesis and invasion

Hepatocyte growth factor/Scatter factor (HGF/SF) is a multifunctional growth factor which can induce diverse biological events. In vitro, these include scattering, invasion, proliferation and branching morphogenesis. In vivo, HGF/SF is responsible for many processes during embryonic development and a variety of activities in adults, and many of these normal activities have been implicated in its role in tumorgenesis and metastasis. The c-Met receptor tyrosine kinase is the only known receptor for HGF/SF and mediates all HGF/SF induced biological activities. Upon HGF/SF stimulation, the c-Met receptor is tyrosine-phosphorylated which is followed by the recruitment of a group of signaling molecules and/or adaptor proteins to its cytoplasmic domain and its multiple docking sites. This action leads to the activation of several different signaling cascades that form a complete network of intra and extracellular responses. Different combinations of signaling pathways and signaling molecules and/or differences in magnitude of responses contribute to these diverse series of HGF/SF-Met induced activities and most certainly are influenced by cell type as well as different cellular environments. In this review, we focus on HGF/SF-induced branching morphogenesis and invasion, and bring together recent new findings which provide insight into how HGF/SF, via c-Met induces this response.     

Listeria InlB takes a different route to met

InlB, a surface protein of the human bacterial pathogen Listeria monocytogenes, interacts with the receptor tyrosine kinase Met on host cells to enable bacterial invasion. In this issue, Niemann et al. (2007) provide the first structural evidence that InlB does not compete for the same interaction site on Met as the natural ligand HGF.     

NK1, a Natural Splice Variant of Hepatocyte Growth Factor/Scatter Factor, Is a Partial Agonist In Vivo

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen, motogen, and morphogen for epithelial cells expressing its tyrosine kinase receptor, the c-met proto-oncogene product, and is required for normal development in the mouse. Inappropriate stimulation of Met signal transduction induces aberrant morphogenesis and oncogenesis in mice and has been implicated in human cancer. NK1 is a naturally occurring HGF/SF splice variant composed of only the amino terminus and first kringle domain. While the biological activities of NK1 have been controversial, in vitro data suggest that it may have therapeutic value as an HGF/SF antagonist. Here, we directly test this hypothesis in vivo by expressing mouse NK1 in transgenic mice and comparing the consequent effects with those observed for mice carrying an HGF/SF transgene. Despite robust expression, NK1 did not behave as an HGF/SF antagonist in vivo. Instead, NK1-transgenic mice displayed most of the phenotypic characteristics associated with HGF/SF-transgenic mice, including enlarged livers, ectopic skeletal-muscle formation, progressive renal disease, aberrant pigment cell localization, precocious mammary lobuloalveolar development, and the appearance of mammary, hepatocellular, and melanocytic tumors. And like HGF/SF-transgenic livers, NK1 livers had higher levels of tyrosine-phosphorylated complexes associated with Met, suggesting that the mechanistic basis for the effects of NK1 overexpression in vivo was autocrine activation of Met. We conclude that NK1 acts in vivo as a partial agonist. As such, the efficacy of NK1 as a therapeutic HGF/SF antagonist must be seriously questioned.     

Shedding-Generated Met Receptor Fragments can be Routed to Either the Proteasomal or the Lysosomal Degradation Pathway

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin-regulated intramembrane proteolysis (PS-RIP) is involved in ligand-independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ-secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N- and C-terminal fragments are degraded directly in the lysosome, with C-terminal fragments escaping γ-secretase cleavage. PS-RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N-terminal fragments associate with the high-affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS-RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand-independent downregulation of the receptor.