ADAM17 is regulated by a rapid and reversible mechanism that controls access to its catalytic site.

Protein ectodomain shedding is crucial for cell-cell interactions because it controls the bioavailability of soluble tumor necrosis factor-α (TNFα) and ligands of the epidermal growth factor (EGF) receptor, and the release of many other membrane proteins. Various stimuli can rapidly trigger ectodomain shedding, yet much remains to be learned about the identity of the enzymes that respond to these stimuli and the mechanisms underlying their activation. Here, we demonstrate that the membrane-anchored metalloproteinase ADAM17, but not ADAM10, is the sheddase that rapidly responds to the physiological signaling pathways stimulated by thrombin, EGF, lysophosphatidic acid and TNFα. Stimulation of ADAM17 is swift and quickly reversible, and does not depend on removal of its inhibitory pro-domain by pro-protein convertases, or on dissociation of an endogenous inhibitor, TIMP3. Moreover, activation of ADAM17 by physiological stimuli requires its transmembrane domain, but not its cytoplasmic domain, arguing against inside-out signaling via cytoplasmic phosphorylation as the underlying mechanism. Finally, experiments with the tight binding hydroxamate inhibitor DPC333, used here to probe the accessibility of the active site of ADAM17, demonstrate that this inhibitor can quickly bind to ADAM17 in stimulated, but not quiescent cells. These findings support the concept that activation of ADAM17 involves a rapid and reversible exposure of its catalytic site.

ADAMs 10 and 17 represent differentially regulated components of a general shedding machinery for membrane proteins such as transforming growth factor alpha, L-selectin, and tumor necrosis factor alpha.

Protein ectodomain shedding is a critical regulator of many membrane proteins, including epidermal growth factor receptor-ligands and tumor necrosis factor (TNF)-alpha, providing a strong incentive to define the responsible sheddases. Previous studies identified ADAM17 as principal sheddase for transforming growth factor (TGF)-alpha and heparin-binding epidermal growth factor, but Ca++ influx activated an additional sheddase for these epidermal growth factor receptor ligands in Adam17-/- cells. Here, we show that Ca++ influx and stimulation of the P2X7R signaling pathway activate ADAM10 as sheddase of many ADAM17 substrates in Adam17-/- fibroblasts and primary B cells. Importantly, although ADAM10 can shed all substrates of ADAM17 tested here in Adam17-/- cells, acute treatment of wild-type cells with a highly selective ADAM17 inhibitor (SP26) showed that ADAM17 is nevertheless the principal sheddase when both ADAMs 10 and 17 are present. However, chronic treatment of wild-type cells with SP26 promoted processing of ADAM17 substrates by ADAM10, thus generating conditions such as in Adam17-/- cells. These results have general implications for understanding the substrate selectivity of two major cellular sheddases, ADAMs 10 and 17.