ADAM17, a prominent member of the ‘Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca2+ elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function.

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, is an important regulator of endothelial cell proliferation and cell survival. The protease controls vital cellular functions through cleavage of growth factors, cytokines and their receptors including transforming growth factor-alpha (TGF-α), tumor necrosis factor-alpha (TNF-α) and TNF-α receptor 1 (TNFR1). TNF-α is the major inducer of endothelial cell death in cardiovascular diseases. The latter are also characterized by elevated plasma and tissue levels of extracellular sphingomyelinase (SMase). Whether the SMase affects ADAM activity and thus endothelial cell function has not been addressed to date. Here, we analyzed the effect of SMase on ADAM17-mediated shedding in COS7 cells and in human umbilical vein endothelial cells (HUVECs). Exposure to SMase significantly increased ADAM17-mediated release of alkaline-phosphatase (AP)-tagged TGF-α in COS7 cells and shedding of endogenously expressed TNFR1 in HUVECs. We previously presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase function. We found that SMase treatment led to PS externalization in both cell types. Transient non-apoptotic PS exposure is often mediated by Ca2+-dependent phospholipid scramblases. Accordingly, the Ca2+-chelator EGTA markedly reduced the breakdown of phospholipid asymmetry and shedding of TGF-α and TNFR1. Moreover, sheddase activity was significantly diminished in the presence of the competing PS-headgroup OPLS. SMase-stimulated TNFR1 shedding strikingly diminished TNF-α-induced signalling cascades and endothelial cell death. Taken together, our data suggest that SMase activity might act as protective factor for endothelial cells in cardiovascular diseases.