Abstract
Ca2+-activated Cl− channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.
Highlights
Disintegrin and metalloproteinases (ADAMs) 10 and 17 can cleave a multitude of membrane-bound proteins in cis to effect release of their ectodomains, a process designated as “shedding”
We report on experiments conducted with ANO1, ANO4, ANO5, ANO7, ANO9, and ANO10 in comparison with ANO6, on the sheddase function of ADAM10 and ADAM17
HEK293T cells were co-transfected with respective anoctamin plasmids and alkalinephosphatase (AP)-tagged ADAM10 (BTC) or ADAM17 substrates (TGFα or AREG)
Summary
Disintegrin and metalloproteinases (ADAMs) 10 and 17 can cleave a multitude of membrane-bound proteins in cis to effect release of their ectodomains, a process designated as “shedding”. ADAM17 promotes inflammation through release of tumor necrosis factor α (TNFα) [1,2,3] and modulates cell function via shedding of epidermal growth factor (EGF) receptor ligands, prominent among which are amphiregulin (AREG), epiregulin, transforming growth factor-alpha (TGFα) and heparin-binding EGF (HB-EGF) [4]. We have recently obtained evidence that plasma membrane dynamics play an important role in controlling sheddase function. Asymmetric distribution of phospholipids in the plasma membrane is a hallmark of the living cell. Externalization of PS is promoted by elevation of intracellular Ca2+, as well as by phorbol esters. We discovered that PS-externalization immediately upregulates ADAM10 and ADAM17 sheddase activity, apparently by promoting the interaction of the catalytic domains of the enzymes with their substrates [15,16]
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