ADAM10 Mediates Ectodomain Shedding of the Betacellulin Precursor Activated by p-Aminophenylmercuric Acetate and Extracellular Calcium Influx

Abstract

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26 -28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase C␦ inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17-and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.

iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding

“…We next tested whether iRhom2 also controls activated shedding of other EGFR ligands (3)(4)(5)27). The PMA-stimulated shedding of the ADAM17 substrates and EGFR ligands amphiregulin and epiregulin (4,5) was also strongly reduced in iRhom2 −/− mEFs compared with WT controls (Fig.1 E and F).…”

“…S1B). Moreover, the ionomycinstimulated shedding of the ADAM10 substrates and EGFR ligands betacellulin (BTC) and EGF (4,15,27) was normal in iRhom2 −/− mEFs ( Fig.1 H and I), arguing against a role of iRhom2 in ADAM10-dependent protein ectodomain shedding.…”

Overexpression of sigma-1 receptor inhibits ADAM10 and ADAM17 mediated shedding in vitro

“…Unlike HB-EGF, Betacellulin (BTC) shedding is mediated by ADAM10 (Sahin et al, 2004) and ionomycin-induced calcium influx can stimulate ADAM10-depedent shedding (Sanderson et al, 2005;Horiuchi et al, 2007). Therefore, we Figure 1.…”

“…Such an effect might be due to the fact that those neurosteriods are sigma-1 receptor agonists (Maurice et al, 2006;Dong et al, 2009;Hayashi and Su, 2010) and activation of the sigma-1 receptor resulted in a bipolar modulation of calcium homeostasis in cell and high concentration of agonist could inhibit the free Ca 2+ in cell (Hayashi et al, 2000;Maurice et al, 2006). Since ADAM10 function is induced by free Ca 2+ in cell (Sanderson et al, 2005;Horiuchi et al, 2007), such inhibition could thus inhibit ADAM10-dependent BTC shedding. On the other hand, although sigma-1 receptor agonists could activate PKC (Fu et al, 2010;Yoon et al, 2010) which can in turn activate ADAM17-dependent shedding (Zheng et al, 2002(Zheng et al, , 2004Sahin et al, 2004), such an effect might be covered by the Figure 3.…”

Regulation of Receptor Tyrosine Kinase Ligand Processing

“…Cite this article as Cold Spring Harb Perspect Biol 2014;6:a008995 bol esters, but can be activated by calcium ionophores, calmodulin inhibition, and the metalloprotease-activating drug p-aminophenylmercuric acetate (Sanderson et al 2005;Horiuchi et al 2007a). More physiologically, activation of purinergic receptors can also trigger ADAM 10 activity (Le Gall et al 2009), as can GPCRs (Lemjabbar and Basbaum 2002).…”