G-protein-coupled receptors (GPCRs) are conventionally considered to function at the plasma membrane, where they detect extracellular ligands and activate heterotrimeric G proteins that transmit ...intracellular signals. Consequently, drug discovery efforts have focused on identification of agonists and antagonists of cell surface GPCRs. However, β-arrestin (ARR)-dependent desensitization and endocytosis rapidly terminate G protein signaling at the plasma membrane. Emerging evidence indicates that GPCRs can continue to signal from endosomes by G-protein- and βARR-dependent processes. By regulating the duration and location of intracellular signaling events, GPCRs in endosomes control critically important processes, including gene transcription and ion channel activity. Thus, GPCRs in endosomes, in addition to at the cell surface, have emerged as important therapeutic targets.
G protein signaling by some GPCRs is not only stimulated at the plasma membrane but also from endosomes after βARR-dependent internalization.
Some GPCRs interact with βARR in a specific conformation, the ‘tail’ conformation, where βARR only is bound to the phosphorylated C-terminal tail and does not block the G-protein-binding site at the intracellular receptor core.
This tail conformation allows GPCRs to interact with G protein and βARR simultaneously to form GPCR–G protein–βARR supercomplexes, which provides a mechanistic basis for endosomal G protein signaling by internalized GPCRs.
Endosomal GPCR signaling plays important cell biological and physiological roles. It was recently found that endosomal GPCR signaling plays a central role in regulating nociception.
Agonists and antagonists can be developed to specifically target endosomal GPCRs to achieve more specific modulation of physiological responses.
G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular ...signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8–37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8–37–cholestanol, but not unconjugated CGRP8–37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8–37–cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund’s adjuvant more effectively than unconjugated CGRP8–37. Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.
Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are ...the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK
R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK
R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK
R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK
R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK
R antagonists. These results reveal a critical role for endosomal signaling of the NK
R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.
Lumbar splanchnic (LSN) and sacral pelvic (PN) nerves convey different mechanosensory information from the colon to the spinal
cord. Here we determined whether these pathways also differ in their ...chemosensitivity and receptor expression. Using an in vitro mouse colon preparation, individual primary afferents were tested with selective P2X and transient receptor potential vanilloid
receptor 1 (TRPV1) receptor ligands. Afferent cell bodies in thoracolumbar and lumbosacral dorsal root ganglia (DRG) were
retrogradely labelled from the colon and analysed for P2X 3 - and TRPV1-like immunoreactivity (LI). Forty per cent of LSN afferents responded to α,β-methylene adenosine 5â²-triphosphate
(α,β-meATP; 1 m m ), an effect that was concentration dependent and reversed by the P2X antagonist pyridoxyl5-phosphate 6-azophenyl-2â²,4â²-disulphonic
acid (PPADS) (100 μ m ). Significantly fewer PN afferents (7%) responded to α,β-meATP. Correspondingly, 36% of colonic thoracolumbar DRG neurones
exhibited P2X 3 -LI compared with only 19% of colonic lumbosacral neurones. Capsaicin (3 μ m ) excited 61% of LSN afferents and 47% of PN afferents; 82% of thoracolumbar and 50% of lumbosacral colonic DRG neurones displayed
TRPV1-LI. Mechanically insensitive afferents were recruited by α,β-meATP or capsaicin, and were almost exclusive to the LSN.
Capsaicin-responsive LSN afferents displayed marked mechanical desensitization after responding to capsaicin, which did not
occur in capsaicin-responsive PN afferents. Therefore, colonic LSN and PN pathways differ in their chemosensitivity to known
noxious stimuli and their corresponding receptor expression. As these pathways relay information that may relate to symptoms
in functional gastrointestinal disease, these results may have implications for the efficacy of therapies targeting receptor
modulation.
Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute ...to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR₂), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR₂-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR₂, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR₂, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR₂-dependent hyperexcitability of nociceptors, and PAR₂ association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR₂. A cholestanol-conjugated PAR₂ antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR₂ signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR₂ antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.
If activation of recombinant G protein-coupled receptors in host cells (by drugs or other ligands) has predictive value, similar data must be obtained with native receptors naturally expressed in ...tissues. Using mouse and human recombinant κ opioid receptors transfected into a host cell, two selectively-acting compounds (ICI204448, asimadoline) equi-effectively activated both receptors, assessed by measuring two different cell signalling pathways which were equally affected without evidence of bias. In mouse intestine, naturally expressing κ receptors within its nervous system, both compounds also equi-effectively activated the receptor, inhibiting nerve-mediated muscle contraction. However, whereas ICI204448 acted similarly in human intestine, where κ receptors are again expressed within its nervous system, asimadoline was inhibitory only at very high concentrations; instead, low concentrations of asimadoline reduced the activity of ICI204448. This demonstration of species-dependence in activation of native, not recombinant κ receptors may be explained by different mouse/human receptor structures affecting receptor expression and/or interactions with intracellular signalling pathways in native environments, to reveal differences in intrinsic efficacy between receptor agonists. These results have profound implications in drug design for κ and perhaps other receptors, in terms of recombinant-to-native receptor translation, species-dependency and possibly, a need to use human, therapeutically-relevant, not surrogate tissues.
The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine
intestine. Multiunit activity was recorded in vitro from mesenteric ...afferents supplying segments of mouse jejunum taken from wild-type (WT) and TRPV1 knockout (TRPV1 â/â ) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so
the pressureâresponse curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase
in activity. Afferent response to distension was significantly lower in TRPV1 â/â than in WT mice. Single-unit analysis revealed three functional types of afferent fibres: (1) low-threshold fibres (2) wide
dynamic range fibres and (3) high-threshold fibres. There was a marked downward shift of the pressureâresponse curve for wide
dynamic range fibres in the TRPV1 â/â mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 m m ) was also attenuated in the TRPV1 â/â mice. In contrast, the response to bath application of bradykinin (1 μ m , 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μ m ) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous
discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1 â/â afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the
results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.
Irritable bowel syndrome (IBS) is a multifactorial condition with principal symptoms of pain and altered bowel function. The kappa-opioid agonist asimadoline is being evaluated in Phase III as a ...potential treatment for IBS. Asimadoline, to date, has shown a good safety profile and the target Phase III population - diarrhea-predominant IBS patients with at least moderate pain - was iteratively determined in a prospective manner from a Phase II dose-ranging study. The clinical data in support of this population are reviewed in this article. Furthermore, the scientific rationale for the use of asimadoline in the treatment of IBS is reviewed. Considering the high patient and societal burdens of IBS, new treatments for IBS represent therapeutic advances.
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