Neurokinin 1 Receptor Endocytosis Research Articles

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of β-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease

Inflammation-induced abnormalities in the subcellular localization and trafficking of the neurokinin 1 receptor in the enteric nervous system.

Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10(-/-)-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions.

Mo1638 Agonist- and Peptidase-Dependent Regulation of Somatostatin Receptor 2A Trafficking in Myenteric Neurons of the Mouse

Background and Aims. Substance P (SP) and the neurokinin 1 receptor (NK1R) control gastrointestinal motility and secretion, and mediate neurogenic inflammation and pain. SPstimulated trafficking regulates NK1R signaling. β-arrestin-mediated NK1R desensitization and endocytosis attenuate plasma membrane signaling, endosomal NK1R signals via βarrestin signalosomes, and NK1R recycling is required for subsequent responses to SP. Since SP and the NK1R mediate intestinal inflammation, we identified neuronal subtypes expressing NK1R and examined the impact of colitis on SP-induced trafficking of the NK1R in enteric neurons. Methods and Results. The neurochemical coding of NK1R-positive neurons of the submucosal and myenteric plexuses of mouse colon was characterized by immunofluorescence. NK1R was expressed by 88% and 22% of submucosal and myenteric neurons, respectively, identified using PGP9.5. NK1R expression was restricted to non-cholinergic secretomotor neurons (NK1R/NPY: 88%; NK1R/VIP: 99%) of the submucosal plexus. In the myenteric plexus, NK1R was localized to intrinsic primary afferent neurons expressing CGRP (76%) and to Dogiel type I neurons containing NOS (9%). Under unstimulated conditions, the NK1R was localized to the plasma membrane of the soma and neurites of myenteric and submucosal neurons. Incubation of colonic explants with SP induced NK1R endocytosis in myenteric and submucosal neurons. The impact of inflammation on NK1R trafficking was examined in IL10 mice treated with piroxicam to induce chronic colitis. In mice with colitis, 55% of NK1R in myenteric neurons was detected in endosomes, compared to 27% in neurons from control mice without colitis (p<0.05). Inflammation-induced NK1R internalization was not detected in submucosal neurons. When explants of myenteric plexus from mice with colitis were incubated in SP-free medium, internalized NK1R recycled to the plasma membrane within 60 min. Subsequent treatment with SP induced NK1R endocytosis. There was no significant difference in the kinetics of SP-induced NK1R endocytosis in myenteric neurons from mice with colitis compared to uninflamed controls. In contrast, NK1R recycling after removal of SP was delayed in myenteric neurons of mice with colitis, with intracellular retention of receptor in inflamed neurons (% NK1R in endosomes at 60 min recovery in SP-free medium: inflamed 32%, control 20%, p<0.05). Conclusions. The NK1R redistributes from the plasma membrane to endosomes of myenteric but not submucosal neurons during colitis. Although SP can evoke NK1R endocytosis in inflamed and uninflamed neurons with similar kinetics, NK1R recycles more slowly in inflamed neurons. During colitis, chronic release of SP and defective recycling machinery, results in intracellular retention of the NK1R, which may transmit inflammatory signals from endosomes. Supported by NIH and NHMRC.

Immunohistochemical localization of substance P NK1 receptor in guinea pig distal colon

Neurokinin receptors facilitate tachykinin mediated intestinal motility and secretion. Distribution of Substance P (SP) neurokinin 1 receptor (NK1r) immunoreactivity (IR) has been previously characterized in guinea pig ileum, but not colon. This study localizes NK1rs in guinea pig distal colon. Neurons were double labelled for NK1r and either acetylcholine transferase (ChAT), calbindin (calb), neuropeptide Y (NPY), nitric oxide synthase (NOS) or SP. The NK1r endocytosis was induced by 10(-5) mol L(-1) SP, septide, [SarMet] SP or neurokinin A. In guinea pig distal colon, NK1r-IR was present on 70% of submucosal neurons. Sixty-threepercent of the NK1r-IR submucosal neurons were ChAT-IR, 16% calb/SP-IR, 19% NPY-IR and 14% NOS-IR neurons. The NK1r-IR was present on 5% of myenteric neurons. Of these 63% were ChAT-IR, 16% calb-IR neurons and 25% NOS-IR. The NK1rs were also on myenteric plexus interstitial cells of Cajal and on circular muscle. In guinea pig distal colon, NK1rs were on 70% of submucosal neurons including all three secretomotor neuron subtypes and sensory neurons, suggesting NK1rs have a major role in neuronal control of mucosal reflexes. The NK1rs were on few myenteric neurons but were dense on muscle cells, suggesting NK1rs affect motility through neuro-muscular rather than neuro-neuronal transmission.

Recycling and Resensitization of the Neurokinin 1 Receptor: INFLUENCE OF AGONIST CONCENTRATION AND Rab GTPASES

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Heterologous regulation of trafficking and signaling of G protein-coupled receptors: beta-arrestin-dependent interactions between neurokinin receptors.

Cells express multiple G protein-coupled receptors that are simultaneously or sequentially activated by agonists. The consequences of activating one receptor on signaling and trafficking of another receptor are unknown. We examined the effects of selective activation of the neurokinin 1 receptor (NK1R) on signaling and trafficking of the NK3R and vice versa. Selective agonists of NK1R and NK3R induced membrane translocation of beta-arrestins (beta-ARRs). Dominant negative beta-ARR(319-418) inhibited endocytosis of NK1R and NK3R. Whereas an NK1R agonist caused sequestration of NK1R with beta-ARR in the same endosomes, thereby depleting them from the cytosol, beta-ARRs did not prominently sequester with the activated NK3R and rapidly returned to the cytosol. In cells coexpressing both receptors, prior activation of the NK1R inhibited endocytosis and homologous desensitization of the NK3R, which was dose-dependently reversed by overexpression of beta-ARR1. Similar results were obtained in enteric neurons that naturally coexpress the NK1R and NK3R. In contrast, activation of the NK3R did not affect NK1R endocytosis or desensitization. Thus, the high-affinity and prolonged interaction of the NK1R with beta-ARRs depletes beta-ARRs from the cytosol and limits their role in desensitization and endocytosis of the NK3R. Because beta-ARRs are critical for desensitization, endocytosis, and mitogenic signaling of many receptors, this sequestration is likely to have important and widespread implications.

Capsaicin vanilloid receptor-1 mediates substance P release in experimental pancreatitis.

We examined whether the capsaicin vanilloid receptor-1 (VR1) mediates substance P (SP) release from primary sensory neurons in experimental pancreatitis. Pancreatitis was achieved by 12 hourly injections of caerulein (50 microg/kg ip) in mice. One group received capsazepine (100 micromol/kg sc), a competitive VR1 antagonist, at 4-h intervals. Neurokinin-1 receptor (NK1R) internalization in acinar cells, used as an index of endogenous SP release, was assessed by immunocytochemical quantification of NK1R endocytosis. The severity of pancreatitis was assessed by measurements of serum amylase, pancreatic myeloperoxidase (MPO) activity, and histological grading. Caerulein administration caused significant elevations in serum amylase and pancreatic MPO activity, produced histological evidence of pancreatitis, and caused a dramatic increase in NK1R endocytosis. Capsazepine treatment significantly reduced the level of NK1R endocytosis, and this was associated with similar reductions in pancreatic MPO activity and histological severity of pancreatitis. These results demonstrate that repeated caerulein stimulation causes experimental pancreatitis that is mediated in part by stimulation of VR1 on primary sensory neurons, resulting in endogenous SP release.

Mechanisms of initiation and termination of signalling by neuropeptide receptors: a comparison with the proteinase-activated receptors

Biological responses to neuropeptides are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell-surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. We have investigated the mechanisms that terminate the proinflammatory effects of the neuropeptide substance P (SP), which are mediated by the neurokinin 1 receptor (NK1R). Neutral endopeptidase degrades SP in the extracellular fluid and is one of the first mechanisms to terminate signalling. G-protein receptor kinases and second-messenger kinases phosphorylate the NK1R to permit interaction with beta-arrestins, which uncouple the receptor from G-proteins to terminate the signal. SP-induces NK1R endocytosis by a beta-arrestin-dependent mechanism, which also involves the GTPases dynamin and Rab5a. Endocytosis contributes to desensitization by depleting receptors from the cell surface. Disruption of these mechanisms results in uncontrolled stimulation and disease. Thus the deletion of neutral endopeptidase in mice exacerbates inflammation of many tissues. There are similarities and distinct differences in the mechanisms that regulate signalling by neuropeptide receptors and other G-protein-coupled receptors, in particular those that are activated irreversibly by proteolysis.

Substance P-induced Trafficking of β-Arrestins: THE ROLE OF β-ARRESTINS IN ENDOCYTOSIS OF THE NEUROKININ-1 RECEPTOR

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.

Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.