Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.signal transduction ͉ trafficking ͉ endocytosis ͉ receptors

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with ␤-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of ␤-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide-receptorarrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK 1R) induced membrane translocation of ␤-arrestins followed by trafficking of the SP-NK1R-␤-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; ␤-arrestins returned to the cytosol, and the NK1R, freed from ␤-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK 1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK 3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptidereceptor-arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control ␤-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.G protein-coupled receptor trafficking ͉ peptidase ͉ resensitization ͉ substance P ͉ inflammation T he mechanisms controlling cellular sensitivity to agonists of G protein-coupled receptors (GPCRs) are crucially important because defects cause uncontrolled signaling and disease, and the molecules involved are therapeutic targets (1). Cell surface peptidases and receptor desensitization terminate cellular responses to neuropeptides, such as substance P (SP). The cell surface metalloendopeptidase neprilysin (NEP; EC 3.4.24.11) degrades SP in the extracellular fluid, which limits neurokinin 1 receptor (NK 1 R) activation and terminates its proinflammatory actions (2-4). G protein receptor kinases (GRKs) phosphorylate agonist-bound NK 1 R to promote interaction with ␤-arrestins, which mediate NK 1 R desensitization and endocytosis and terminate NK 1 R signaling (5).In contrast to the termination of GPCR signaling, little is known about GPCR recycling and resensitization, which mediate sustained signaling. The affinity of interaction between GPCRs and ␤-arrestins d...

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

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Calcitonin gene-related peptide (CGRP) 2 belongs to the calcitonin family of regulatory peptides and is produced by tissuespecific alternate splicing of transcripts from the calcitonin gene (1). CGRP is a potent vasodilator and mediator of neurogenic inflammation and pain transmission (2, 3). Notably, CGRP has a causative role in migraine headaches and is thus a mediator of human disease (4). In view of the importance of CGRP in health and disease, it is of interest to understand the mechanisms that control cellular responses to this peptide.Unusually for neuropeptides, the CGRP receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a G-protein-coupled receptor (GPCR) that shares 55% amino acid sequence identity with the calcitonin receptor (5), whereas RAMP1, RAMP2, and RAMP3 are single transmembrane proteins with ϳ30% identity (6). CLR functions as either a CGRP receptor, when coexpressed with RAMP1, or an adrenomedullin receptor, when coexpressed with RAMP2 or -3 (6); the extracellular domain of the RAMP imparts this specificity (7,8). When expressed alone, CLR is retained in the endoplasmic reticulum (6), and RAMP1 is retained in the endoplasmic reticulum and the Golgi apparatus (9 -11). However, when coexpressed, CLR and RAMP1 traffic to the plasma membrane. Thus, RAMP1 is also a chaperone that targets CLR to the plasma membrane. However, it remains to be determined if CLR and RAMP1 are invariably associated after receptor activation.Upon activation with CGRP, CLR, but not RAMP1, is phosphorylated and interacts with ␤-arrestins (12). ␤-Arrestins are adapters for clathrin and AP2, and the CLR, RAMP1, and ␤-arrestin complex undergoes dynamin-dependent endocytosis in clathrin-coated pits by well defined mechanisms (11, 12). However, little is known about the mechanisms of post-endocytic sorting of GPCRs, such as CLR, or accessory proteins, such as RAMP1, to degradative or recycling pathways. It is important to understand the post-endocytic sorting of CLR and RAMP1, because recycling may permit rapid resensitization of CGRP signaling, whereas degradation would prevent sustained, uncontrolled CGRP signaling during conditions of sustained peptide release.␤-Arrestins can influence the rate of recycling of GPCRs. "Class A" receptors (e.g. ␤ 2 adrenergic receptor (␤ 2 AR), -opi-* This work was supported by National Institutes of Health Grants DK39957 (to N. W. B.), DK43207 (to N. W. B.), DK57840 (to N. W. B.), and DK52388 (to E. F. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. RAMP1, receptor activity-modifying protein; NK 1 R, neurokinin 1 receptor; ␤ 2 AR, ␤ 2 adrenergic receptor; PAR 2 , protease-activated receptor 2; LAMP1, lysosomal-associated glycomembrane protein 1; EEA1, early endosomal antigen 1; BSA, bovine serum albumin; HA, hemagglutinin; ...

Background. Extracorporeal membrane oxygenation (ECMO) artificially supports respiratory and cardiac function when conventional techniques fail. ECMO has been described as a treatment modality for acute pulmonary and cardiac failure following orthotopic liver transplantation (OLT). Here, we present a series of adult OLT recipients placed on ECMO after transplantation for both respiratory and cardiac indications and review the literature on the role of ECMO in the setting of OLT. Methods. For the patient series, we cross-referenced all patients who underwent OLT at our institution between 2007 and 2018 with the ECMO database of our institution and described these cases. For the literature review, we identified cases and series that described the use of ECMO after liver transplantation in adult recipients. Results. A total of 1792 patients underwent OLT. Eight patients were placed on ECMO (0.4%), 5 men and 3 women aged 28 to 68 years (4 venovenous and 4 venoarterial). Three of (38%) 8 patients survived to discharge and are alive today. In the literature, we identified 3 series and 12 case reports of ECMO following OLT, with the majority of the literature derived from the Asian OLT experience. Conclusions. ECMO following liver transplantation should be considered as a viable rescue strategy in patients with severe cardiopulmonary failure. ECMO is particularly effective if the cause of cardiopulmonary failure is recognized promptly and is thought to be transient. This is the largest series in the United States and demonstrates a 38% survival rate, which is comparable to other reports in the literature from Asia.

Agonist-induced trafficking of G-protein-coupled receptors (GPCRs) 2 between the plasma membrane and organelles determines cellular responsiveness. The molecular mechanisms of receptor endocytosis have been thoroughly investigated. Many activated GPCRs exemplified by the angiotensin II type 1A receptor, ␤ 2 -adrenergic receptor, neurokinin-1 receptor (NK 1 R), and protease-activated receptor-1 and -2 (PAR 2 ) are phosphorylated by G-protein receptor kinases (1-5). This phosphorylation increases the affinity of receptors for ␤-arrestins, which translocate to the receptors at the plasma membrane (3, 6 -10). ␤-Arrestins (a) sterically hinder the interaction between GPCRs and heterotrimeric G-proteins to desensitize G-protein signaling (3,11,12); (b) are adaptor proteins for clathrin and activator protein-2 and are thus required for endocytosis of GPCRs (3,7,12,13); and (c) serve as molecular scaffolds for the formation of signaling modules that include components of the MAPK (mitogen-activated protein kinase) cascade such as Src, Raf-1, MEKK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase), and activated ERK1/2 (extracellular signal-regulated kinase-1 and -2) (14 -17). However, in comparison with endocytosis, much less is known about the molecular mechanisms of post-endocytic sorting of GPCRs back to the plasma membrane (recycling) or to lysosomes or proteasomes (down-regulation).The nature of interaction between GPCRs and ␤-arrestins determines the rate of receptor recycling. GPCRs can be divided into two classes according to their interaction with ␤-arrestins (18). "Class A" receptors (e.g. ␤ 2 -adrenergic, -opioid, ␣ 1b -adrenergic, and neurokinin-3 receptors) show a preference for ␤-arrestin-2 over ␤-arrestin-1 and interact with low affinity and transiently with ␤-arrestin-2 to rapidly dissociate and recycle (18 -20). "Class B" receptors (e.g. NK 1 R, angiotensin II type 1A receptor, and neurotensin receptor-1) form high affinity and sustained interactions with both isoforms of ␤-arrestin and then slowly recycle to the plasma membrane (18). The existence of Ser and Thr residues within the C-terminal domains of these receptors, which are sites for phosphorylation by G-protein receptor kinases, specifies high affinity interactions with ␤-arrestins and therefore determines the rates of

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