Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY 10021, USA.
J Cell Sci. 2010 Nov 15;123(Pt 22):3913-22. doi: 10.1242/jcs.069997. Epub 2010 Oct 27.
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.
蛋白细胞外结构域脱落对于细胞间相互作用至关重要，因为它控制着可溶性肿瘤坏死因子-α（TNFα）和表皮生长因子（EGF）受体配体的生物利用度，以及许多其他膜蛋白的释放。各种刺激可以迅速触发细胞外结构域脱落，但对于响应这些刺激的酶的身份以及它们激活的机制，仍有许多需要了解。在这里，我们证明膜锚定金属蛋白酶 ADAM17 （而不是 ADAM10）是快速响应凝血酶、EGF、溶血磷脂酸和 TNFα 等生理信号通路刺激的脱落酶。ADAM17 的刺激迅速且可快速逆转，并且不依赖于蛋白转化酶去除其抑制性前结构域，也不依赖于内源性抑制剂 TIMP3 的解离。此外，生理刺激激活 ADAM17 需要其跨膜结构域，但不需要其细胞质结构域，这表明通过细胞质磷酸化的内信号传递不是潜在机制。最后，使用紧密结合的羟肟酸抑制剂 DPC333 进行的实验，用于探测 ADAM17 的活性位点的可及性，证明该抑制剂可以快速结合到受刺激而不是静止的细胞中的 ADAM17。这些发现支持 ADAM17 激活涉及其催化位点的快速和可逆暴露的概念。
