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

Abstract

SummaryProtein ectodomain shedding is crucial for cell-cell interactions because it controls the bioavailability of soluble tumor necrosis factora (TNFa) 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 TNFa. 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.

Roles of extracellular nucleotides and P2 receptors in ectodomain shedding

“…In the absence of ADAM17, ADAM10 is able to mediated BzATP-induced CD62L shedding [39,40]. However BzATP-stimulated CD62L shedding mediated by ADAM17 was significantly more rapid than shedding mediated by ADAM10 [39], corroborating a role for ADAM17 as the principal sheddase. Such studies highlight the complexity in ectodomain shedding, often revealing dominant but redundant roles for many sheddases.…”

“…Subsequently, a role for ADAM17 in this process was shown in murine B cells and T cells [39,40]. In the absence of ADAM17, ADAM10 is able to mediated BzATP-induced CD62L shedding [39,40]. However BzATP-stimulated CD62L shedding mediated by ADAM17 was significantly more rapid than shedding mediated by ADAM10 [39], corroborating a role for ADAM17 as the principal sheddase.…”

“…and oxidised ATP, impaired this nucleotide-induced CD23 shedding [34], confirming a role for P2X7 in this process. Since then, P2X7-induced CD23 shedding has been described for human monocyte-derived dendritic cells [35], human monocyte-derived Langerhans cells [36], human RPMI 8226 multiple myeloma B cells [37], human B cells [38] and murine B cells [38,39].…”

“…Second, the hydroxamic acid-based protease inhibitor of Zn 2+ -dependent metalloprotease, Ro 31-9790, inhibited P2X7-induced CD23 shedding more potently than P2X7-induced CD62L shedding. A role for ADAM10 in ATP-or BzATP-induced CD23 shedding was first shown in human histiocytic lymphoma U937 cells using an inhibitory prodomain construct of ADAM10, A10-(23-213) [43], and subsequently in CD23-transfected CHO cells and murine B cells using GI 254023X [39,40].…”

“…A metalloproteases was first implicated in this process when Ro 31-9790 was shown to inhibit P2X7-induced CD62L shedding in CLL cells [34]. Subsequently, a role for ADAM17 in this process was shown in murine B cells and T cells [39,40]. In the absence of ADAM17, ADAM10 is able to mediated BzATP-induced CD62L shedding [39,40].…”