An Arginine Stretch Limits ADAM10 Exit from the Endoplasmic Reticulum

Abstract

A disintegrin and metalloproteinase 10 (ADAM10) is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a number of proteins implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease. ADAM10 is synthesized in an inactive form, which is proteolytically activated during its forward transport along the secretory pathway and at the plasma membrane. Therefore, modulation of its trafficking could provide a mechanism to finely tune its shedding activity. Here we report the identification of an endoplasmic reticulum (ER) retention motif within the ADAM10 intracellular C-terminal tail. Sequential deletion/mutagenesis analyses showed that an arginine-rich ( 723 RRR) sequence was responsible for the retention of ADAM10 in the ER and its inefficient surface trafficking. Mutating the second arginine to alanine was sufficient to allow ER exit and surface expression in both heterologous cells and hippocampal neurons. As synapse-associated protein 97 (SAP97) binds ADAM10 at its cytoplasmic tail and facilitates forward ADAM10 trafficking in neurons, we tested whether SAP97 could modulate ER export. However, neither expression nor Ser-39 phosphorylation of SAP97 in heterologous cells or hippocampal neurons were sufficient to allow the ER exit of ADAM10, suggesting that other signaling pathways or alternative binding partners are responsible for ADAM10 ER exit. Together, these results identify a novel mechanism regulating the intracellular trafficking and membrane delivery of ADAM10.A disintegrin and metalloproteinase 10 (ADAM10) 2 belongs to a large family of membrane-anchored metalloproteases, which are known as the ADAM protein family. ADAMs mediate the proteolytic cleavage of transmembrane proteins in their juxtamembrane region, causing their shedding, i.e. the release of their extracellular domain in a soluble form. In addition, through the intracellularly retained stubs, ADAMs can initiate the activation of intracellular signaling cascades. Because of their metalloprotease, integrin binding, cell adhesion, and signaling functions, ADAMs are well positioned to coordinate cellular processes that are required for neural development, plasticity, and repair (1-3).ADAM10 works as a sheddase for a large number of transmembrane proteins involved in a variety of biological functions, and has been implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease (AD) (4, 5). Because of these links, much effort is currently directed toward developing tools which modulate ADAM10 activity and can be used to target these pathologies.ADAM10 is a multidomain transmembrane glycoprotein which is expressed ubiquitously (6). It contains an N-terminal signal sequence followed by a prodomain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, an EGFlike repeat, a transmembrane domain and a SH3-binding cytoplasmic tail (7). ADAM10 is synthesized in an inactive form that carries a proprotein convertase (PC) recognition sequence between the prodomain and the c...

The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

“…Our results demonstrate that the cytoplasmic domain of ADAM10 functions as a negative regulator of the constitutive activity of ADAM10 but is dispensable for the rapid post-translational stimulation of this enzyme. The cytoplasmic domain of ADAM10 is known to contain an ER retention motif and basolateral sorting signals in polarized epithelial cells (20,26). We found that deletion of the ER retention motif or a point mutation in this motif both had the same effect as deleting the cytoplasmic domain altogether.…”

“…2C). 723 RAR (A10 RRR3 RAR) or its deletion through a truncation after residue 721 (ADAM10⌬721, A10⌬721) has been shown to increase transport of the mutant proteins to the cell surface (20). Here, we demonstrate that both mutants have a similarly increased constitutive activity over 2 h as ADAM10⌬cyto (Fig.…”

“…We found that deletion of the ER retention motif or a point mutation in this motif both had the same effect as deleting the cytoplasmic domain altogether. Both mutants have been shown to traffic to the cell surface more efficiently (20), and we provide evidence that this is also the case for the ADAM10⌬cyto protein. The lack of an ER retention motif also explains our finding that the levels of proADAM10⌬cyto are lower than those of pro-ADAM10 WT, despite transfection with comparable amounts of expression vector for either form.…”

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

“…An important question that arises from the current study concerns the mechanism by which the TspanC8 tetraspanins promote trafficking of ADAM10 to the cell surface. ADAM10 was recently shown to possess an argininebased endoplasmic reticulum (ER) retention motif that prevents efficient trafficking of the metalloprotease to the cell surface (42). However, it was not clear from that study how the retention motif is overcome and ADAM10 is released from the ER.…”

Ectodomain shedding and ADAMs in development

“…Development 139, 3693-3709 (2012) Interestingly, at steady state, the vast majority of endogenous ADAM10 and ADAM17 is localized in the endoplasmic reticulum (ER) and Golgi apparatus and little protein is observed at the plasma membrane where ectodomain shedding occurs (Schlondorff et al, 2000; Gutwein et al, 2003). For ADAM10, an ER-retention motif within the ADAM10 intracellular C-terminal tail is also observed (Marcello et al, 2010), and the binding of proteins such as tetraspanins in the case of ADAM10 (Prox et al, 2012) and iRHOM2 in the case of ADAM17 (Adrain et al, 2012;McIlwain et al, 2012) can trigger the export of these proteases from the endoplasmic reticulum. The binding of iRHOM2 (also known as RHBDF2) to ADAM17 also contributes to innate immunity and pathogen defense (McIlwain et al, 2012).…”