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...

Release of cytokines, growth factors and other life-essential molecules from precursors by a-disintegrin-and-metalloproteases (ADAMs) is regulated with high substrate-specificity. We hypothesized that this is achieved by cleavage-regulatory intracellular-domain (ICD)-modifications of the precursors. We show here that cleavage-stimuli-induced specific ICD-modifications cause structural substrate changes that enhance ectodomain sensitivity of neuregulin-1 (NRG1; epidermal-growth-factor) or CD44 (receptor-tyrosine-kinase (RTK) co-receptor) to chymotrypsin/trypsin or soluble ADAM. This inside-out signal transfer required substrate homodimerization and was prevented by cleavage-inhibitory ICD-mutations. In chimeras, regulation could be conferred to a foreign ectodomain, suggesting a common higher-order structure. We predict that substrate-specific protease-accessibility-regulation controls release of numerous ADAM substrates.