Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.epidermal growth factor receptor | transactivation T he ectodomains of many cell surface proteins are shed from the surface (i.e., "ectodomain shedding") by metalloproteases. Ectodomain shedding generates many diverse bioactive cytokines and growth factors, and governs important cellular processes in the developing and adult organism, including the control of growth, adhesion, and motility of cells (reviewed in refs. 1-3). EGF receptor activation generates signals for cell proliferation, migration, differentiation, or survival. The 12 EGF family members are synthesized as cell surface transmembrane precursors. The active growth factors are released by A disintegrin and metalloproteinases (ADAMs) and activate specific heterodimeric EGF receptors on the cell surface connected to diverse intracellular signaling pathways (4, 5). Increased shedding of EGF ligands has been linked to different clinical pathologic processes (6-10); hence, therapeutic control of ligand release would be beneficial. Of the 12 functional ADAMs encoded in the human genome (3) only two-ADAM10 and ADAM17-handle most of the numerous ADAM substrates, in particular, the EGF ligands. However, broad-spectrum metalloprotease inhibitors tested for clinical use have failed as a result of indiscriminate blockade of substrate cleavage, leading to clinical side effects (11). Even recently developed selective ADAM inhibitors still affect the cleavage of many substrates (12). Modulation of the release of specific ADAM substrates has been impossible to date because it is unknown how cleavage specificity is regulated on the molecular level. It is therefore necessary to identify key signals that determine substrate specificity of cleavage.Ectodomain cleavage is made specific by a number of intracellular signals; e.g., by calcium influx, by activation of G protein-coup...

Summary Background  Matrix metalloproteinases (MMPs) contribute to matrix remodelling in venous leg ulcers. Extracellular MMP inducer (EMMPRIN; CD147) has been reported to increase MMP expression, and membrane type 1 MMP (MT1‐MMP) has been implicated in the activation of MMPs. Objectives  To examine whether and to what degree EMMPRIN, MMP‐2, MT1‐MMP and membrane type 2 MMP (MT2‐MMP) are expressed in venous leg ulcers as well as the association with MMP activity. Methods  EMMPRIN, MMP‐2, MT1‐MMP and MT2‐MMP were analysed by zymography and immunohistochemistry in biopsies from healthy skin and lesional tissue from venous leg ulcers. Results  Zymography provided direct evidence of increased proteolytic activity of MMP‐2 in lesional skin in comparison with healthy controls. Immunostaining showed intense expression of EMMPRIN, MMP‐2, MT1‐MMP and MT2‐MMP in dermal structures of venous leg ulcers, whereas only EMMPRIN and MMP‐2 showed elevated expression in perivascular regions. Our findings indicate that venous leg ulcers are characterized by elevated expression of EMMPRIN, MMP‐2, MT1‐MMP and MT2‐MMP. The immunohistological findings of skin alterations reflect the dynamic process of activation of soluble and membrane‐bound MMPs, which may be highly induced by EMMPRIN. Conclusions  These data suggest for the first time that membrane‐bound MMPs may favour enhanced turnover of the extracellular matrix and support unrestrained MMP activity in venous leg ulcers.

The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGF␣, neuregulin 1␤ (NRG), and heparinbinding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substratespecific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGF␣ and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKC enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.Metalloprotease-mediated ectodomain cleavage of transmembrane proteins such as EGF ligand precursors is involved in the regulation of many physiological signaling pathways, and its dysregulation can cause kidney disease, heart disease, and cancer (1, 2). For example, in the kidney, many of the deleterious effects of chronic exposure to the G-protein-coupled receptor (GPCR) 2 agonist angiotensin II are mediated by metalloproteinase (ADAM17)-dependent release of the EGF ligand TGF␣ and subsequent epidermal growth factor receptor activation. Angiotensin II is the main effector of the renin-angiotensin system and has important roles in the regulation of blood pressure and aldosterone secretion (3).Although several ADAM gene disruptions are lethal in the mouse, metalloprotease inhibitors have shown therapeutic potential; however, not unexpectedly, the essential role of ADAMs in normal physiology as well as the broad spectrum nature of currently available inhibitors are probably the cause for a number of their side effects. In addition, concerns have been raised that some metalloproteases act as tumor suppressors (4,5). A more precise inhibiti...

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Background: Intracellular domain (ICD) modifications regulate extracellular ectodomain cleavage by metalloproteases. How this inside-out signal is relayed is unknown. Results: Cleavage requires substrate homodimerization; ICD modifications likely induce a relative positional change of the dimerization partners, allowing cleavage. Conclusion: Substrate dimerization might be a general requirement for cleavage. Significance: Our results fill an important gap in understanding growth factor release by ectodomain cleavage.

Ectodomain cleavage (shedding) of transmembrane proteins by metalloproteases (MMP) generates numerous essential signaling molecules, but its regulation is not totally understood. CD44, a cleaved transmembrane glycoprotein, exerts both antiproliferative or tumor-promoting functions, but whether proteolysis is required for this is not certain. CD44-mediated contact inhibition and cellular proliferation are regulated by counteracting CD44 C-terminal interacting proteins, the tumor suppressor protein merlin (NF2) and ERM proteins (ezrin, radixin, moesin). We show here that activation or overexpression of constitutively active merlin or downregulation of ERMs inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced [as well as serum, hepatocyte growth factor (HGF), or plateletderived growth factor (PDGF)] CD44 cleavage by the metalloprotease ADAM10, whereas overexpressed ERM proteins promoted cleavage. Merlin-and ERM-modulated Ras or Rac activity was not required for this function. However, latrunculin (an actin-disrupting toxin) or an ezrin mutant which is unable to link CD44 to actin, inhibited CD44 cleavage, identifying a cytoskeletal C-terminal link as essential for induced CD44 cleavage. Cellular migration, an important tumor property, depended on CD44 and its cleavage and was inhibited by merlin. These data reveal a novel function of merlin and suggest that CD44 cleavage products play a tumor-promoting role. Neuregulin, an EGF ligand released by ADAM17 from its proform NRG1, is predominantly involved in regulating cellular differentiation. In contrast to CD44, release of neuregulin from its pro-form was not regulated by merlin or ERM proteins. Disruption of the actin cytoskeleton however, also inhibited NRG1 cleavage. This current study presents one of the first examples of substrate-selective cleavage regulation.

bEctodomain cleavage by A-disintegrin and -metalloproteases (ADAMs) releases many important biologically active substrates and is therefore tightly controlled. Part of the regulation occurs on the level of the enzymes and affects their cell surface abundance and catalytic activity. ADAM-dependent proteolysis occurs outside the plasma membrane but is mostly controlled by intracellular signals. However, the intracellular domains (ICDs) of ADAM10 and -17 can be removed without consequences for induced cleavage, and so far it is unclear how intracellular signals address cleavage. We therefore explored whether substrates themselves could be chosen for proteolysis via ICD modification. We report here that CD44 (ADAM10 substrate), a receptor tyrosine kinase (RTK) coreceptor required for cellular migration, and pro-NRG1 (ADAM17 substrate), which releases the epidermal growth factor (EGF) ligand neuregulin required for axonal outgrowth and myelination, are indeed posttranslationally modified at their ICDs. Tetradecanoyl phorbol acetate (TPA)-induced CD44 cleavage requires dephosphorylation of ICD serine 291, while induced neuregulin release depends on the phosphorylation of several NRG1-ICD serines, in part mediated by protein kinase C␦ (PKC␦). Downregulation of PKC␦ inhibits neuregulin release and reduces ex vivo neurite outgrowth and myelination of trigeminal ganglion explants. Our results suggest that specific selection among numerous substrates of a given ADAM is determined by ICD modification of the substrate. Many transmembrane proteins on the cell surface are subject to proteolytic cleavage of their ectodomains, predominantly by metalloproteases (ectodomain shedding) (1-3). Ectodomain shedding regulates numerous important molecules involved in signal transfer between the extracellular space and the cell's interior and thus influences many cellular functions (1, 3). This includes, for example, the biological availability of epidermal growth factor (EGF) receptor ligands such as neuregulin (NRG1) (4, 5) and the modulation of complex cellular phenotypes required for contact inhibition of cells involving the hyaluronic acid receptor CD44 (4). NRG1 regulates neurite outgrowth and myelination but also has important functions in the development of other organs, for instance, the heart (6-9). When bound to hyaluronan, CD44 triggers a proliferation-inhibitory pathway (10-12). On the other hand, cancer stem cells carry CD44 (13-15), and, in this context, CD44 promotes tumor growth and metastasis (16-21), likely via alternative splice forms of CD44 that act as growth factor-enriching coreceptors for receptor tyrosine kinases (RTKs) (22,23).Inappropriate proteolysis of a number of shed substrates is associated with diseases when cleavage is either upregulated or reduced (24, 25). Equally, total knockout of substrates leads to significant phenotypes (26,27). This indicates that ectodomain cleavage requires tight regulation. How ectodomain cleavage is regulated and made substrate specific is largely unknown to date.The metallop...

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin‐CD44 complex through loss of Merlin, unleashes putative tumor‐ or metastasis‐promoting functions of CD44. To evaluate the relevance of the Merlin‐CD44 interaction in vivo, we compared tumor growth and progression in Cd44‐positive and Cd44‐negative Nf2‐mutant mice. Heterozygous Nf2‐mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2‐mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4β1 (very late antigen‐4, VLA‐4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA‐4 β1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis‐promoting role in the absence of Merlin.