Endosomal Endothelin-converting Enzyme-1

Abstract

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and ␤-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. ␤-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, ␤-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls ␤-arrestin-dependent signaling of endocytosed GPCRs.Signal transduction at the plasma membrane is tightly regulated by well characterized mechanisms. For the large family of G protein-coupled receptors (GPCRs), 2 regulation of extracellular agonist concentration and receptor coupling to heterotrimeric G proteins control signal transduction. Cell-surface metalloendopeptidases, exemplified by neprilysin, degrade neuropeptides in the extracellular fluid to regulate receptor activation (1, 2). G protein receptor kinases phosphorylate activated GPCRs to promote their interaction with ␤-arrestins, which uncouple receptors from G proteins and mediate desensitization (3). However, activated GPCRs internalize, and endocytosed receptors continue to signal by G protein-independent mechanisms. ␤-Arrestins mediate endocytosis and intracellular signaling of GPCRs. ␤-Arrestins couple GPCRs to clathrin and AP2 to mediate endocytosis (4, 5) and are scaffolds that recruit Src, Raf-1, and mitogen-activated protein kinases (MAPKs) to GPCRs in endosomes forming a MAPK signalosome, which determines the location and function of activated extracellular signal-regulated kinases (ERKs) (6 -9). Compared with our understanding of receptor regulation at the plasma membrane, little is known about the mechanisms that regulate GPCR signaling and trafficking at the endosomal membrane. Specifically, it is not known whether agonist interaction with GPCRs or GPCR interaction with ␤-arrestins is necessary for receptor signaling in endosomes. Regulation of these interactions could determine the duration of ␤-arrestin-mediated ERK activation, with important functional implications (10, 11). We hypothesized that mechanisms that regulate GPCRs at the plasma membrane also control receptor signaling and trafficking at the endosomal membrane. We recently reported t...

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

“…With respect to these, it is interesting to remark that a member of M13 family (ECE-1) localizes into the luminal side of endosomal membrane where it degrades interna lized neuropeptides (e.g., substance P, apelin-13) (El Messari et al , 2004 ;Cottrell et al , 2009 ), suggesting their possible biological relevance as substrates of this type of enzymes. As a matter of fact, besides the classical involvement of neuropeptides in neurotransmission and vasal smooth muscle cells constriction, at least for some of them, a novel role in the modulation of immune responses has been envisaged (Skidgel et al , 1984 ;Johnson et al , 1999 ;Marriott and Bost , 2001 ;Yaraee et al , 2007 ), affecting proinfl ammatory cytokine and chemokine activation (in the case of apelin-13) and macrophage activation (in the case of the substance P) in the early phases of immune responses to Salmonella infection (Kincy -Cain and Bost, 1996 ;O ' Connor et al, 2004 ;Leeper et al , 2009 ).…”

“…In this respect, it is noteworthy that degradation of neuropeptides by ECE-1 in endosomes under acidic conditions (i.e., pH 5.5) is documented (Fahnoe et al, 2000) and it is associated to intracellular signaling and receptor turnover (El Messari et al , 2004 ;Roostermann et al, 2007;Cottrell et al , 2009 ); analogously, we found that Zmp1 optimum activity toward the synthetic substrate is at pH 6.3 (Figure 2A), a value corresponding to that of the early endosome (Pethe et al , 2004 ). Therefore, it might be speculated that, similarly to ECE-1, Zmp1 operates within the phagosome, arresting its maturation (Master et al , 2008 ;Johansen et al , 2011 ).…”

Regulation of Membrane Cholecystokinin-2 Receptor by Agonists Enables Classification of Partial Agonists as Biased Agonists

“…Although fluorescent CCK or gastrin remained trapped in endocytosic vesicles over times as long as 6 h, we do not know if the peptidic ligands were still intact and bound to internalized CCK2R at these times or if they were degraded by proteases. Other studies have shown that substance P, calcitonin gene-related peptide, and somatostatin are degraded in acidified early endosomes following internalization with their cognate receptors (32). Interestingly, it was suggested that retention of ligand integrity or ligand degradation may impact signaling triggered by internalized GPCR (33).…”

Endosomal Deubiquitinating Enzymes Control Ubiquitination and Down-regulation of Protease-activated Receptor 2

“…Endothelin-converting enzyme-1 degrades substance P and calcitonin gene-related peptide in acidified endosomes to disrupt the ligand-GPCR-␤-arrestin complex, which promotes ␤-arrestin return to the cytosol and receptor recycling to the plasma membrane (47,48). This mechanism also attenuates substance P-induced ERK activation (35). Although the ligand for PAR 2 is tethered to the receptor, other endosomal proteases may degrade the ligand to disrupt the PAR 2 -␤-arrestin-MAPK complex and thereby attenuate ERK signaling.…”