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

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In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2–4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)–dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

KChIPs (K+ channel interacting proteins) regulate the function of A-type Kv4 potassium channels by modifying channel properties and by increasing their cell surface expression. We have explored factors affecting the localisation of Kv4.2 and the targeting of KChIP1 and other NCS proteins by using GFP-variant fusion proteins expressed in HeLa cells. ECFP-Kv4.2 expressed alone was not retained in the ER but reached the Golgi complex. In cells co-expressing ECFP-Kv4.2 and KChIP1-EYFP, the two proteins were co-localised and were mainly present on the plasma membrane. When KChIP1-EYFP was expressed alone it was instead targeted to punctate structures. This was distinct from the localisation of the NCS proteins NCS-1 and hippocalcin, which were targeted to the trans-Golgi network (TGN) and plasma membrane. The membrane localisation of each NCS protein required myristoylation and minimal myristoylation motifs of hippocalcin or KChIP1 were sufficient to target fusion proteins to either TGN/plasma membrane or to punctate structures. The existence of targeting information within the N-terminal motifs was confirmed by mutagenesis of residues corresponding to three conserved basic amino acids in hippocalcin and NCS-1 at positions 3, 7 and 9. Residues at these positions determined intracellular targeting to the different organelles. Myristoylation and correct targeting of KChIP1 was required for the efficient traffic of ECFP-Kv4.2 to the plasma membrane. Expression of KChIP1(1-11)-EYFP resulted in the formation of enlarged structures that were positive for ERGIC-53 and β-COP. ECFP-Kv4.2 was also accumulated in these structures suggesting that KChIP1(1-11)-EYFP inhibited traffic out of the ERGIC. We suggest that KChIP1 is targeted by its myristoylation motif to post-ER transport vesicles where it could interact with and regulate the traffic of Kv4 channels to the plasma membrane under the influence of localised Ca2+ signals.

The E3 ubiquitin ligase c-Cbl ubiquitinates the G proteincoupled receptor protease-activated receptor 2 (PAR 2 ), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR 2 deubiquitination and its importance in trafficking and signaling of endocytosed PAR 2 are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR 2 ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR 2 . Trapping PAR 2 in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR 2 with ␤-arrestin2 or the duration of PAR 2 -induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR 2 but not for regulating PAR 2 dissociation from ␤-arrestin2 or PAR 2 -mediated ERK2 activation. Ubiquitination of certain G protein-coupled receptors (GPCRs)3 is an essential signal for their postendocytic trafficking to lysosomes, which prevents uncontrolled signaling during chronic stimulation. Agonists stimulate ubiquitination of the ␤ 2 -adrenergic receptor (␤ 2 AR), chemokine (CXC motif) receptor 4, and protease-activated receptor 2 (PAR 2 ), and the E3 ubiquitin ligases that mediate ubiquitination of these GPCRs and associated proteins, such as ␤-arrestins, have been identified (1-3). Although ubiquitination of these receptors is not required for endocytosis, ubiquitin-resistant mutant receptors show diminished postendocytic sorting to lysosomes and impaired down-regulation. However, despite of the reversible nature of this post-translational modification, little is known about the role of deubiquitinating proteases (DUBs) in the postendocytic trafficking and signaling of GPCRs.Our understanding of the role of DUBs in postendocytic receptor trafficking mostly derives from studies of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Two endosomal DUBs, AMSH (associated molecule with the Src homology 3 domain of STAM (signal-transducing adapter molecule)) and UBPY (ubiquitin-specific protease Y) (also known as USP8), regulate deubiquitination and postendocytic trafficking of EGFR (4). AMSH belongs to the JAMM (JAB1/MPN/Mov34) family of metalloproteases and shows specificity for Lys 63 -over Lys 48 -linked ubiquitin chains (5, 6). UBPY is a cysteine protease of the ubiquitin-specific protease (USP) family and does not discriminate between Lys 48 -and Lys 63 -linked ubiquitin (7,8). Activated EGFR recruits the E3 ligase c-Cbl, which ubiquitinates the receptor (9). Ubiquitinated EGFR then interacts with the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate)-ST...

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR 2 ), which is required for post-endocytic sorting of PAR 2 to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR 2 . In HEK-PAR 2 cells, PAR 2 activating peptide (PAR 2 -AP) induced PAR 2 trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR 2 and c-Cbl accumulated, and PAR 2 failed to traffic to lysosomes. Overexpression of HRS prevented PAR 2 -AP-induced degradation of PAR 2 , as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR 2 in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR 2 ⌬14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR 2 ⌬14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR 2 and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and nonubiquitinated PAR 2 and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS. Many G protein-coupled receptors (GPCRs)2 are rapidly endocytosed after agonist binding, but the pathway of postendocytic trafficking depends on the receptor and the nature of the stimulus. Some GPCRs are sorted to lysosomes or proteasomes, where degradation irrevocably inactivates internalized receptors and prevents uncontrolled signaling during chronic stimulation (1-4). Other GPCRs recycle to the plasma membrane, which mediates resensitization of signal transduction (5-8). Our understanding of the mechanisms underlying the critical sorting of GPCRs to these divergent pathways, degradative or recycling, is incomplete and controversial. Ubiquitination of certain receptors, exemplified by protease-activated receptor 2 (PAR 2 ) and the chemokine (C-X-C motif) receptor 4 (CXCR4), serves as a signal for sorting receptors into a lysosomal or proteasomal degradation pathway (4, 9 -12). However, other receptors, including the ␦-opioid receptor and calcitonin receptor-like receptor (CLR) (8, 13,14), traffic to the degradative pathway by ubiquitin-independent mechanisms. It is important to elucidate the sorting machinery that diverts a rec...

Urocortins (UCNs) and their receptors are potent immunoregulators in the gastrointestinal (GI) tract, where they can exert both pro- and anti-inflammatory effects. We examined the contribution of Ucn1 and its receptors to the pathogenesis, progression, and resolution of colitis. Trinitrobenzene sulfonic acid was used to induce colitis in rats. Ucn1 mRNA and immunoreactivity (IR) were ubiquitously expressed throughout the GI tract under basal conditions. During colitis, Ucn1 mRNA levels fell below basal levels on day 1 then increased again by day 6, in association with an increase in the number of Ucn1-IR inflammatory cells. Ucn1-IR cells were also numerous in proliferating granulation tissue. In contrast to Ucn1 expression, average phosphorylated ERK1/2 (pERK1/2) expression rose above controls levels on day 1 and was very low on day 6 of colitis. Knockdown of corticotropin-releasing factor 2 (CRF(2)) but not CRF(1) by RNA interference during colitis significantly decreased the macroscopic lateral spread of ulceration compared with uninjected controls or animals with CRF(1) knockdown. After knockdown of CRF(2), but not of CRF(1) during colitis, edema resolution assessed microscopically was slowed, and myeloperoxidase activity remained elevated even at day 6. Ucn1 and TNF-α mRNA peaked earlier, whereas pERK1/2 activation was attenuated after CRF(2) knockdown. Thus we conclude that local CRF(2) and pERK1/2 activation is pivotal for macroscopic spread of colitis and resolution of edema. Elimination of CRF(2), but not CRF(1), results in uncoordinated immune and pERK1/2 signaling responses.