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Ectodomain 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 Cδ (PKCδ). Downregulation of PKCδ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many life-essential molecules such as growth factors, cytokines, ectoenzymes, and decoy receptors are produced by ectodomain cleavage of transmembrane precursor molecules. Not surprisingly, misregulation of such essential functions is linked to numerous diseases. Ectodomain cleavage is the function of transmembrane ADAMs (a disintegrin and metalloprotease) and other membrane-bound metalloproteases, which have an extracellular catalytic domain. Almost all work on ectodomain cleavage regulation has focused on the control of enzyme activity determined by substrate cleavage as surrogate. However, the number of substrates far exceeds the number of enzymes. Specificity can therefore not be achieved by solely modulating enzyme activity. Here, we argue that specific regulatory pathways must exist to control the availability and susceptibility of substrates. [ABSTRACT FROM AUTHOR]

Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-α, is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage-- hypertonic stress, phorbol ester, or activation of G-protein- coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS) -based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hyper tonic stress led to strong cleavage of HBO-EEG and UNRIG but only moderate cleavage of TAG-α. HBO-EEG, UNRIG, and TAG-α cleavage was not dependent on PC, and only HBO-EEG and UNRIG cleavage were inhibited by BB 94. In contrast, hobo 12-prostate-13-acetate (PA) -induced cleavage of I-IBM-EEG, UNRIG, and TAG-α was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-&alpha: and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different extracellular stimuli into ectodomain cleavage. [ABSTRACT FROM AUTHOR]

Ectodomain shedding of transmembrane precursor proteins generates numerous life-essential molecules, such as epidermal growth factor receptor ligands. This cleavage not only releases the regulatory growth factor, but it is also the required first step for the subsequent processing by γ-secretase and the release of gene regulatory intracellular fragments. Signaling within the cell modifies the cytoplasmic tails of substrates, a step important in starting the specific and regulated cleavage of a large number of studied substrates. Ectodomain cleavage occurs, however, on the outside of the plasma membrane and is carried out by membrane-bound metalloproteases. How the intracellular domain modification communicates with the ectodomain of the substrate to allow for cleavage to occur is unknown. Here, we show that homodimerization of a cluster-of-differentiation-44 or of pro-neuregulin-1 monomers represents an essential precondition for their regulated ectodomain cleavage. Both substrates are associated with their respective metalloproteases under both basal or cleavage-stimulated conditions. These interactions only turn productive by specific intracellular signal- induced intracellular domain modifications of the substrates, which in turn regulate metalloprotease access to the substrates' ectodomain and cleavage.Wepropose that substrate intracellular domain modification induces a relative rotation or other positional change of the dimerization partners that allow metalloprotease cleavage in the extracellular space. Our findings fill an important gap in understanding substrate-specific inside-out signal transfer along cleaved transmembrane proteins and suggest that substrate dimerization (homo- or possibly heterodimerization) might represent a general principle in ectodomain shedding. [ABSTRACT FROM AUTHOR]

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 TGFα, neuregulin 1β (NRG), and heparinbinding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FA SEB I.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific 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, TGFα and heparm-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 PKCζ 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. [ABSTRACT FROM AUTHOR]