Anoctamin-6 regulates ADAM sheddase function

Abstract ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates including TGF-alpha, Amphiregulin (AREG) and TNF-Receptor 1 (TNFR1). We recently presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. Anoctamin-6 (ANO6) has Ca 2+ -dependent phospholipid scramblase activity and it followed that the functions of ANO6 and ADAM17 might be linked. We report that overexpression of ANO6 in HEK293T cells led to increased Ca 2+ -mediated PS-exposure that was indeed accompanied by enhanced release of AREG and TGF-alpha. The effect was not observed when cells were treated with the PKC-dependent ADAM17 activator PMA. Transformation of cells with a constitutively active ANO6 mutant led to spontaneous PS-exposure and to the release of ADAM17-substrates in the absence of any stimuli. Inhibitor experiments indicated that ANO6-mediated enhancement of substrate cleavage simultaneously broadened the spectrum of participating metalloproteinases. In complementary experiments, siRNA-mediated downregulation of ANO6 was shown to decrease ionophore-mediated release of TNFR1 in human umbilical vein endothelial cells (HUVECs). We conclude that ANO6, by virtue of its scramblase activity, may play a role as an important regulator of the ADAM-network in the plasma membrane.