β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

Recently, a requirement for β-arrestin–mediated endocytosis in the activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) by several G protein–coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Gαq-coupled proteinase–activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, β-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2δST363/6A), which is unable to interact with β-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(δST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, β-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists

Agonist-induced endocytosis of rat somatostatin receptor 1

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand

Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia

Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2

Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists

Activation of pruritogenic TGR5, MRGPRA3, and MRGPRC11 on colon-innervating afferents induces visceral hypersensitivity

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MRGPRA3, or MRGPRC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an "itch cocktail" augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MRGPRX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders. - 2019 American Society for Clinical Investigation. All rights reserved.Work was supported by a National Health and Medical Research Council of Australia (NHMRC) Project Grant (1083480 to SMB and DPP), an NHMRC R.D. Wright Biomedical Research Fellow (APP1126378 to SMB), and an Australian Research Council (ARC) Discovery Early Career Research Award (DE130100223 to AMH). NWB was supported by grants from the NIH (NS102722; DE026806; DK118971) and the US Department of Defence (W81XWH1810431).Scopu

TGR5 agonists induce peripheral and central hypersensitivity to bladder distension

The mechanisms underlying chronic bladder conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder syndrome (OAB) are incompletely understood. However, targeting specific receptors mediating neuronal sensitivity to specific stimuli is an emerging treatment strategy. Recently, irritant-sensing receptors including the bile acid receptor TGR5, have been identified within the viscera and are thought to play a key role in neuronal hypersensitivity. Here, in mice, we identify mRNA expression of TGR5 (Gpbar1) in all layers of the bladder as well as in the lumbosacral dorsal root ganglia (DRG) and in isolated bladder-innervating DRG neurons. In bladderinnervating DRG neurons Gpbar1 mRNA was 100% co-expressed with Trpv1 and 30% co-expressed with Trpa1. In vitro live-cell calcium imaging of bladder-innervating DRG neurons showed direct activation of a sub-population of bladder-innervating DRG neurons with the synthetic TGR5 agonist CCDC, which was diminished in Trpv1−/− but not Trpa1−/− DRG neurons. CCDC also activated a small percentage of non-neuronal cells. Using an ex vivo mouse bladder afferent recording preparation we show intravesical application of endogenous (5α-pregnan-3β-ol-20-one sulphate, Pg5α) and synthetic (CCDC) TGR5 agonists enhanced afferent mechanosensitivity to bladder distension. Correspondingly, in vivo intravesical administration of CCDC increased the number of spinal dorsal horn neurons that were activated by bladder distension. The enhanced mechanosensitivity induced by CCDC ex vivo and in vivo was absent using Gpbar1−/− mice. Together, these results indicate a role for the TGR5 receptor in mediating bladder afferent hypersensitivity to distension and thus may be important to the symptoms associated with IC/BPS and OAB.Ashlee Caldwell, Luke Grundy, Andrea M. Harrington, Sonia Garcia, Caraballo, Joel Castro, Nigel W. Bunnett, Stuart M. Brierle

Prognostic and Mechanistic Potential of Progesterone Sulfates in Intrahepatic Cholestasis of Pregnancy and Pruritus Gravidarum

A challenge in obstetrics is to distinguish pathological symptoms from those associated with normal changes of pregnancy, typified by the need to differentiate whether gestational pruritus of the skin is an early symptom of intrahepatic cholestasis of pregnancy (ICP) or due to benign pruritus gravidarum. ICP is characterized by raised serum bile acids and complicated by spontaneous preterm labor and stillbirth. A biomarker for ICP would be invaluable for early diagnosis and treatment and to enable its differentiation from other maternal diseases. Three progesterone sulfate compounds, whose concentrations have not previously been studied, were newly synthesized and assayed in the serum of three groups of ICP patients and found to be significantly higher in ICP at 9-15 weeks of gestation and prior to symptom onset (group 1 cases/samples: ICP n = 35/80, uncomplicated pregnancy = 29/100), demonstrating that all three progesterone sulfates are prognostic for ICP. Concentrations of progesterone sulfates were associated with itch severity and, in combination with autotaxin, distinguished pregnant women with itch that would subsequently develop ICP from pruritus gravidarum (group 2: ICP n = 41, pruritus gravidarum n = 14). In a third group of first-trimester samples all progesterone sulfates were significantly elevated in serum from low-risk asymptomatic women who subsequently developed ICP (ICP/uncomplicated pregnancy n = 54/51). Finally, we show mechanistically that progesterone sulfates mediate itch by evoking a Tgr5-dependent scratch response in mice. Conclusion: Our discovery that sulfated progesterone metabolites are a prognostic indicator for ICP will help predict onset of ICP and distinguish it from benign pruritus gravidarum, enabling targeted obstetric care to a high-risk population. Delineation of a progesterone sulfate-TGR5 pruritus axis identifies a therapeutic target for itch management in ICP

Apparent Alkyl Transfer and Phenazine Formation via an Aryne Intermediate

Treatment of chlorotriaryl derivatives 3a and 3d or fluorotriaryl derivatives 3b and 3e with potassium diisopropylamide afforded alkyl-shifted phenazine derivatives 5a/5b, rather than the expected 9-membered triazaorthocyclophane 2a. The phenazine derivatives were isolated in 78–98% yield depending on the halogen and alkyl group present. In the absence of the halogen (chloro or fluoro), the apparent alkyl shift proceeds more slowly and cannot proceed via the intermediacy of the aryne intermediate. Mechanistic possibilities include intramolecular nucleophilic attack on an aryne intermediate leading to a zwitterionic intermediate and alkyl transfer via a 5-endo-tet process, or via a Smiles rearrangement

Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

<p>Abstract</p> <p>Background</p> <p>Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade.</p> <p>Methods</p> <p>Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV) was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF₂α production. Proliferation assays were also performed in presence of different prostaglandins (PGs).</p> <p>Results</p> <p>Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor <it>in vivo </it>in skeletal muscle cells and in satellite cells and <it>in vitro </it>in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-^12,14-prostaglandin J₂(15Δ-PGJ₂), a product of COX-2-derived prostaglandin D₂, stimulated myoblast proliferation, but not PGE₂and PGF₂α.</p> <p>Conclusions</p> <p>Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream activation of COX-2.</p