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1
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Multimodal Analgesia in the Aesthetic Plastic Surgery: Concepts and Strategies. Plast Reconstr Surg Glob Open 2022; 10:e4310. [PMID: 35572190 PMCID: PMC9094416 DOI: 10.1097/gox.0000000000004310] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2022] [Accepted: 03/17/2022] [Indexed: 12/13/2022]
Abstract
Postoperative pain management is crucial for aesthetic plastic surgery procedures. Poorly controlled postoperative pain results in negative physiologic effects and can affect length of stay and patient satisfaction. In light of the growing opioid epidemic, plastic surgeons must be keenly familiar with opioid-sparing multimodal analgesia regimens to optimize postoperative pain control. Methods A review study based on multimodal analgesia was conducted. Results We present an overview of pain management strategies pertaining to aesthetic plastic surgery and offer a multimodal analgesia model for outpatient aesthetic surgery practices. Conclusion This review article presents an evidence-based approach to multimodal pain management for aesthetic plastic surgery.
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2
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Oberhauser L, Stoeber M. Biosensors Monitor Ligand-Selective Effects at Kappa Opioid Receptors. Handb Exp Pharmacol 2021; 271:65-82. [PMID: 33387066 DOI: 10.1007/164_2020_427] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to β-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor-biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.
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Affiliation(s)
- Lucie Oberhauser
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Miriam Stoeber
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
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3
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Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies. Sci Rep 2020; 10:8585. [PMID: 32444688 PMCID: PMC7244497 DOI: 10.1038/s41598-020-65589-7] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2019] [Accepted: 05/05/2020] [Indexed: 01/08/2023] Open
Abstract
The δ-opioid receptor (DOP) is an attractive pharmacological target due to its potent analgesic, anxiolytic and anti-depressant activity in chronic pain models. However, some but not all selective DOP agonists also produce severe adverse effects such as seizures. Thus, the development of novel agonists requires a profound understanding of their effects on DOP phosphorylation, post-activation signaling and dephosphorylation. Here we show that agonist-induced DOP phosphorylation at threonine 361 (T361) and serine 363 (S363) proceeds with a temporal hierarchy, with S363 as primary site of phosphorylation. This phosphorylation is mediated by G protein-coupled receptor kinases 2 and 3 (GRK2/3) followed by DOP endocytosis and desensitization. DOP dephosphorylation occurs within minutes and is predominantly mediated by protein phosphatases (PP) 1α and 1β. A comparison of structurally diverse DOP agonists and clinically used opioids demonstrated high correlation between G protein-dependent signaling efficacies and receptor internalization. In vivo, DOP agonists induce receptor phosphorylation in a dose-dependent and agonist-selective manner that could be blocked by naltrexone in DOP-eGFP mice. Together, our studies provide novel tools and insights for ligand-activated DOP signaling in vitro and in vivo and suggest that DOP agonist efficacies may determine receptor post-activation signaling.
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4
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Quirion B, Bergeron F, Blais V, Gendron L. The Delta-Opioid Receptor; a Target for the Treatment of Pain. Front Mol Neurosci 2020; 13:52. [PMID: 32431594 PMCID: PMC7214757 DOI: 10.3389/fnmol.2020.00052] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Accepted: 03/13/2020] [Indexed: 12/15/2022] Open
Abstract
Nowadays, pain represents one of the most important societal burdens. Current treatments are, however, too often ineffective and/or accompanied by debilitating unwanted effects for patients dealing with chronic pain. Indeed, the prototypical opioid morphine, as many other strong analgesics, shows harmful unwanted effects including respiratory depression and constipation, and also produces tolerance, physical dependence, and addiction. The urgency to develop novel treatments against pain while minimizing adverse effects is therefore crucial. Over the years, the delta-opioid receptor (DOP) has emerged as a promising target for the development of new pain therapies. Indeed, targeting DOP to treat chronic pain represents a timely alternative to existing drugs, given the weak unwanted effects spectrum of DOP agonists. Here, we review the current knowledge supporting a role for DOP and its agonists for the treatment of pain. More specifically, we will focus on the cellular and subcellular localization of DOP in the nervous system. We will also discuss in further detail the molecular and cellular mechanisms involved in controlling the cellular trafficking of DOP, known to differ significantly from most G protein-coupled receptors. This review article will allow a better understanding of how DOP represents a promising target to develop new treatments for pain management as well as where we stand as of our ability to control its cellular trafficking and cell surface expression.
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Affiliation(s)
- Béatrice Quirion
- Faculté de Médecine et des Sciences de la Santé, Département de Pharmacologie-Physiologie, Institut de Pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Francis Bergeron
- Faculté de Médecine et des Sciences de la Santé, Département de Pharmacologie-Physiologie, Institut de Pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Véronique Blais
- Faculté de Médecine et des Sciences de la Santé, Département de Pharmacologie-Physiologie, Institut de Pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Louis Gendron
- Faculté de Médecine et des Sciences de la Santé, Département de Pharmacologie-Physiologie, Institut de Pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada
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5
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Stoeber M, Jullié D, Li J, Chakraborty S, Majumdar S, Lambert NA, Manglik A, von Zastrow M. Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells. eLife 2020; 9:54208. [PMID: 32096468 PMCID: PMC7041944 DOI: 10.7554/elife.54208] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2019] [Accepted: 01/29/2020] [Indexed: 12/21/2022] Open
Abstract
G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric ‘bias’ into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists. About a third of all drugs work by targeting a group of proteins known as G-protein coupled receptors, or GPCRs for short. These receptors are found on the surface of cells and transmit messages across the cell’s outer barrier. When a signaling molecule, like a hormone, is released in the body, it binds to a GPCR and changes the receptor’s shape. The change in structure affects how the GPCR interacts and binds to other proteins on the inside of the cell, triggering a series of reactions that alter the cell’s activity. Scientists have previously seen that a GPCR can trigger different responses depending on which signaling molecule is binding on the surface of the cell. However, the mechanism for this is unknown. One hypothesis is that different signaling molecules change the GPCR’s preference for binding to different proteins on the inside of the cell. The challenge has been to observe this happening without interfering with the process. Stoeber et al. have now tested this idea by attaching fluorescent tags to proteins that bind to activated GPCRs directly and without binding other signaling proteins. This meant these proteins could be tracked under a microscope as they made their way to bind to the GPCRs. Stoeber et al. focused on one particular GPCR, known as the opioid receptor, and tested the binding of two different opioid signaling molecules, etorphine and Dynorphin A. The experiments revealed that the different opioids did affect which of the engineered proteins would preferentially bind to the opioid receptor. This was followed by a similar experiment, where the engineered proteins were replaced with another protein called GRK2, which binds to the opioid receptor under normal conditions in the cell. This showed that GRK2 binds much more strongly to the opioid receptor when Dynorphin A is added compared to adding etorphine. These findings show that GPCRs can not only communicate that a signaling molecule is binding but can respond differently to convey what molecule it is more specifically. This could be important in developing drugs, particularly to specifically trigger the desired response and reduce side effects. Stoeber et al. suggest that an important next step for research is to understand how the GPCRs preferentially bind to different proteins.
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Affiliation(s)
- Miriam Stoeber
- Department of Psychiatry, University of California, San Francisco, San Francisco, United States.,Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland
| | - Damien Jullié
- Department of Psychiatry, University of California, San Francisco, San Francisco, United States.,Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
| | - Joy Li
- Department of Psychiatry, University of California, San Francisco, San Francisco, United States.,Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
| | - Soumen Chakraborty
- Center for Clinical Pharmacology, Washington University School of Medicine, St. Louis, United States.,St Louis College of Pharmacy, St. Louis, United States
| | - Susruta Majumdar
- Center for Clinical Pharmacology, Washington University School of Medicine, St. Louis, United States.,St Louis College of Pharmacy, St. Louis, United States
| | - Nevin A Lambert
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, United States
| | - Aashish Manglik
- Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States.,Department of Anesthesia, University of California, San Francisco, San Francisco, United States
| | - Mark von Zastrow
- Department of Psychiatry, University of California, San Francisco, San Francisco, United States.,Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
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6
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Caputi FF, Rullo L, Stamatakos S, Candeletti S, Romualdi P. Interplay between the Endogenous Opioid System and Proteasome Complex: Beyond Signaling. Int J Mol Sci 2019; 20:ijms20061441. [PMID: 30901925 PMCID: PMC6470665 DOI: 10.3390/ijms20061441] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Revised: 03/13/2019] [Accepted: 03/19/2019] [Indexed: 02/07/2023] Open
Abstract
Intracellular signaling mechanisms underlying the opioid system regulation of nociception, neurotransmitters release, stress responses, depression, and the modulation of reward circuitry have been investigated from different points of view. The presence of the ubiquitin proteasome system (UPS) in the synaptic terminations suggest a potential role of ubiquitin-dependent mechanisms in the control of the membrane occupancy by G protein-coupled receptors (GPCRs), including those belonging to the opioid family. In this review, we focused our attention on the role played by the ubiquitination processes and by UPS in the modulation of opioid receptor signaling and in pathological conditions involving the endogenous opioid system. The collective evidence here reported highlights the potential usefulness of proteasome inhibitors in neuropathic pain, addictive behavior, and analgesia since these molecules can reduce pain behavioral signs, heroin self-administration, and the development of morphine analgesic tolerance. Moreover, the complex mechanisms involved in the effects induced by opioid agonists binding to their receptors include the ubiquitination process as a post-translational modification which plays a relevant role in receptor trafficking and degradation. Hence, UPS modulation may offer novel opportunities to control the balance between therapeutic versus adverse effects evoked by opioid receptor activation, thus, representing a promising druggable target.
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Affiliation(s)
- Francesca Felicia Caputi
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Irnerio 48, 40126 Bologna, Italy.
| | - Laura Rullo
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Irnerio 48, 40126 Bologna, Italy.
| | - Serena Stamatakos
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Irnerio 48, 40126 Bologna, Italy.
| | - Sanzio Candeletti
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Irnerio 48, 40126 Bologna, Italy.
| | - Patrizia Romualdi
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Irnerio 48, 40126 Bologna, Italy.
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7
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Abstract
BACKGROUND It has been demonstrated that κ-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the κ-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how κ-opioid receptors respond to hypoxia and reoxygenation. METHODS Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor-tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. RESULTS Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced κ-opioid receptor internalization. CONCLUSIONS Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.
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8
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Gendron L, Cahill CM, von Zastrow M, Schiller PW, Pineyro G. Molecular Pharmacology of δ-Opioid Receptors. Pharmacol Rev 2016; 68:631-700. [PMID: 27343248 PMCID: PMC4931872 DOI: 10.1124/pr.114.008979] [Citation(s) in RCA: 89] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Opioids are among the most effective analgesics available and are the first choice in the treatment of acute severe pain. However, partial efficacy, a tendency to produce tolerance, and a host of ill-tolerated side effects make clinically available opioids less effective in the management of chronic pain syndromes. Given that most therapeutic opioids produce their actions via µ-opioid receptors (MOPrs), other targets are constantly being explored, among which δ-opioid receptors (DOPrs) are being increasingly considered as promising alternatives. This review addresses DOPrs from the perspective of cellular and molecular determinants of their pharmacological diversity. Thus, DOPr ligands are examined in terms of structural and functional variety, DOPrs' capacity to engage a multiplicity of canonical and noncanonical G protein-dependent responses is surveyed, and evidence supporting ligand-specific signaling and regulation is analyzed. Pharmacological DOPr subtypes are examined in light of the ability of DOPr to organize into multimeric arrays and to adopt multiple active conformations as well as differences in ligand kinetics. Current knowledge on DOPr targeting to the membrane is examined as a means of understanding how these receptors are especially active in chronic pain management. Insight into cellular and molecular mechanisms of pharmacological diversity should guide the rational design of more effective, longer-lasting, and better-tolerated opioid analgesics for chronic pain management.
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Affiliation(s)
- Louis Gendron
- Département de Pharmacologie-Physiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke, Centre d'excellence en neurosciences de l'Univeristé de Sherbrooke, and Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada (L.G.); Québec Pain Research Network, Sherbrooke, Quebec, Canada (L.G.); Departments of Anesthesiology and Perioperative Care and Pharmacology, University of California, Irvine, California (C.M.C.); Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada (C.M.C.); Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California (M.v.Z.); Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montréal, Montreal, Quebec, Canada (P.W.S.); and Departments of Psychiatry, Pharmacology, and Neurosciences, Faculty of Medicine, University of Montréal and Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada (G.P.)
| | - Catherine M Cahill
- Département de Pharmacologie-Physiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke, Centre d'excellence en neurosciences de l'Univeristé de Sherbrooke, and Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada (L.G.); Québec Pain Research Network, Sherbrooke, Quebec, Canada (L.G.); Departments of Anesthesiology and Perioperative Care and Pharmacology, University of California, Irvine, California (C.M.C.); Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada (C.M.C.); Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California (M.v.Z.); Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montréal, Montreal, Quebec, Canada (P.W.S.); and Departments of Psychiatry, Pharmacology, and Neurosciences, Faculty of Medicine, University of Montréal and Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada (G.P.)
| | - Mark von Zastrow
- Département de Pharmacologie-Physiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke, Centre d'excellence en neurosciences de l'Univeristé de Sherbrooke, and Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada (L.G.); Québec Pain Research Network, Sherbrooke, Quebec, Canada (L.G.); Departments of Anesthesiology and Perioperative Care and Pharmacology, University of California, Irvine, California (C.M.C.); Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada (C.M.C.); Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California (M.v.Z.); Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montréal, Montreal, Quebec, Canada (P.W.S.); and Departments of Psychiatry, Pharmacology, and Neurosciences, Faculty of Medicine, University of Montréal and Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada (G.P.)
| | - Peter W Schiller
- Département de Pharmacologie-Physiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke, Centre d'excellence en neurosciences de l'Univeristé de Sherbrooke, and Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada (L.G.); Québec Pain Research Network, Sherbrooke, Quebec, Canada (L.G.); Departments of Anesthesiology and Perioperative Care and Pharmacology, University of California, Irvine, California (C.M.C.); Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada (C.M.C.); Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California (M.v.Z.); Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montréal, Montreal, Quebec, Canada (P.W.S.); and Departments of Psychiatry, Pharmacology, and Neurosciences, Faculty of Medicine, University of Montréal and Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada (G.P.)
| | - Graciela Pineyro
- Département de Pharmacologie-Physiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke, Centre d'excellence en neurosciences de l'Univeristé de Sherbrooke, and Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada (L.G.); Québec Pain Research Network, Sherbrooke, Quebec, Canada (L.G.); Departments of Anesthesiology and Perioperative Care and Pharmacology, University of California, Irvine, California (C.M.C.); Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada (C.M.C.); Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California (M.v.Z.); Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montréal, Montreal, Quebec, Canada (P.W.S.); and Departments of Psychiatry, Pharmacology, and Neurosciences, Faculty of Medicine, University of Montréal and Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada (G.P.)
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Zhang Z, Zhang W, Huang S, Sun Q, Wang Y, Hu Y, Sun N, Zhang Y, Jiang Z, Minato N, Pin JP, Su L, Liu J. GABAB receptor promotes its own surface expression by recruiting a Rap1-dependent signaling cascade. J Cell Sci 2015; 128:2302-13. [DOI: 10.1242/jcs.167056] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2014] [Accepted: 05/05/2015] [Indexed: 12/11/2022] Open
Abstract
ABSTRACT
G-protein-coupled receptors (GPCRs) are key players in cell signaling, and their cell surface expression is tightly regulated. For many GPCRs such as β2-AR (β2-adrenergic receptor), receptor activation leads to downregulation of receptor surface expression, a phenomenon that has been extensively characterized. By contrast, some other GPCRs, such as GABAB receptor, remain relatively stable at the cell surface even after prolonged agonist treatment; however, the underlying mechanisms are unclear. Here, we identify the small GTPase Rap1 as a key regulator for promoting GABAB receptor surface expression. Agonist stimulation of GABAB receptor signals through Gαi/o to inhibit Rap1GAPII (also known as Rap1GAP1b, an isoform of Rap1GAP1), thereby activating Rap1 (which has two isoforms, Rap1a and Rap1b) in cultured cerebellar granule neurons (CGNs). The active form of Rap1 is then recruited to GABAB receptor through physical interactions in CGNs. This Rap1-dependent signaling cascade promotes GABAB receptor surface expression by stimulating receptor recycling. Our results uncover a new mechanism regulating GPCR surface expression and also provide a potential explanation for the slow, long-lasting inhibitory action of GABA neurotransmitter.
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Affiliation(s)
- Zongyong Zhang
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Wenhua Zhang
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Siluo Huang
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Qian Sun
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Yunyun Wang
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Yongjian Hu
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Ninghua Sun
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Yilei Zhang
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Zhihua Jiang
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Nagahiro Minato
- Department of Immunology and Cell Biology, Kyoto University, Kyoto 606-8501, Japan
| | - Jean-Philippe Pin
- Institut de Génomique Fonctionnelle, CNRS, UMR 5203, Université Montpellier 1 et 2, Montpellier cedex 5 34094, France
| | - Li Su
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Jianfeng Liu
- Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan 430074, China
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10
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Mudgal A, Pasha S. Role of opioid receptor heterodimerization in pain modulation and tolerance development. World J Pharmacol 2015; 4:144-159. [DOI: 10.5497/wjp.v4.i1.144] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2014] [Revised: 10/09/2014] [Accepted: 02/11/2015] [Indexed: 02/06/2023] Open
Abstract
Protein to protein interactions leading to homo/heteromerization of receptor is well documented in literature. These interactions leading to dimeric/oligomers formation of receptors are known to modulate their function, particularly in case of G-protein coupled receptors. The opioid receptor heteromers having changed pharmacological properties than the constituent protomers provides preferences for novel drug targets that could lead to potential analgesic activity devoid of tolerance and physical dependence. Heterodimerization of opioid receptors appears to generate novel binding properties with improved specificity and lack of side effects. Further the molecules which can interact simultaneously to both the protomers of the heteromer, or to both the binding sites (orthosteric and allosteric) of a receptor protein could be potential therapeutic molecules. This review highlights the recent advancements in exploring the plausible role of heteromerization of opioid receptors in induction of tolerance free antinociception.
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11
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Solinski HJ, Gudermann T, Breit A. Pharmacology and signaling of MAS-related G protein-coupled receptors. Pharmacol Rev 2014; 66:570-97. [PMID: 24867890 DOI: 10.1124/pr.113.008425] [Citation(s) in RCA: 104] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Signaling by heptahelical G protein-coupled receptors (GPCR) regulates many vital body functions. Consequently, dysfunction of GPCR signaling leads to pathologic states, and approximately 30% of all modern clinical drugs target GPCR. One decade ago, an entire new GPCR family was discovered, which was recently named MAS-related G protein-coupled receptors (MRGPR) by the HUGO Gene Nomenclature Committee. The MRGPR family consists of ∼40 members that are grouped into nine distinct subfamilies (MRGPRA to -H and -X) and are predominantly expressed in primary sensory neurons and mast cells. All members are formally still considered "orphan" by the Committee on Receptor Nomenclature and Drug Classification of the International Union of Basic and Clinical Pharmacology. However, several distinct peptides and amino acids are discussed as potential ligands, including β-alanine, angiotensin-(1-7), alamandine, GABA, cortistatin-14, and cleavage products of proenkephalin, pro-opiomelanocortin, prodynorphin, or proneuropeptide-FF-A. The full spectrum of biologic roles of all MRGPR is still ill-defined, but there is evidence pointing to a role of distinct MRGPR subtypes in nociception, pruritus, sleep, cell proliferation, circulation, and mast cell degranulation. This review article summarizes findings published in the last 10 years on the phylogenetic relationships, pharmacology, signaling, physiology, and agonist-promoted regulation of all MRGPR subfamilies. Furthermore, we highlight interactions between MRGPR and other hormonal systems, paying particular attention to receptor multimerization and morphine tolerance. Finally, we discuss the challenges the field faces presently and emphasize future directions of research.
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Affiliation(s)
- Hans Jürgen Solinski
- Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, Munich, Germany
| | - Thomas Gudermann
- Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, Munich, Germany
| | - Andreas Breit
- Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, Munich, Germany
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Gendron L, Mittal N, Beaudry H, Walwyn W. Recent advances on the δ opioid receptor: from trafficking to function. Br J Pharmacol 2014; 172:403-19. [PMID: 24665909 DOI: 10.1111/bph.12706] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2013] [Revised: 03/17/2014] [Accepted: 03/18/2014] [Indexed: 01/12/2023] Open
Abstract
UNLABELLED Within the opioid family of receptors, δ (DOPrs) and μ opioid receptors (MOPrs) are typical GPCRs that activate canonical second-messenger signalling cascades to influence diverse cellular functions in neuronal and non-neuronal cell types. These receptors activate well-known pathways to influence ion channel function and pathways such as the map kinase cascade, AC and PI3K. In addition new information regarding opioid receptor-interacting proteins, downstream signalling pathways and resultant functional effects has recently come to light. In this review, we will examine these novel findings focusing on the DOPr and, in doing so, will contrast and compare DOPrs with MOPrs in terms of differences and similarities in function, signalling pathways, distribution and interactions. We will also discuss and clarify issues that have recently surfaced regarding the expression and function of DOPrs in different cell types and analgesia. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.
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Affiliation(s)
- Louis Gendron
- Département de physiologie et biophysique, Institut de pharmacologie de Sherbrooke, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada
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13
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Dimers of G-protein coupled receptors as versatile storage and response units. Int J Mol Sci 2014; 15:4856-77. [PMID: 24651459 PMCID: PMC3975428 DOI: 10.3390/ijms15034856] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2014] [Revised: 02/28/2014] [Accepted: 03/04/2014] [Indexed: 11/17/2022] Open
Abstract
The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.
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14
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Nooh MM, Chumpia MM, Hamilton TB, Bahouth SW. Sorting of β1-adrenergic receptors is mediated by pathways that are either dependent on or independent of type I PDZ, protein kinase A (PKA), and SAP97. J Biol Chem 2013; 289:2277-94. [PMID: 24324269 DOI: 10.1074/jbc.m113.513481] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The β1-adrenergic receptor (β1-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension. Recycling of agonist-internalized β1-AR is dependent on type I PSD-95/DLG/ZO1 (PDZ) in the C-tail of the β1-AR and on protein kinase A (PKA) activity (Gardner, L. A., Naren, A. P., and Bahouth, S. W. (2007) J. Biol. Chem. 282, 5085-5099). We explored the effects of point mutations in the PDZ and in the activity of PKA on recycling of the β1-AR and its binding to the PDZ-binding protein SAP97. These studies indicated that β1-AR recycling was inhibited by PKA inhibitors and by mutations in the PDZ that interfered with SAP97 binding. The trafficking effects of short sequences differing in PDZ and SAP97 binding were examined using chimeric mutant β1-AR. β1-AR chimera containing the type I PDZ of the β2-adrenergic receptor that does not bind to SAP97 failed to recycle except when serine 312 was mutated to aspartic acid. β1-AR chimera with type I PDZ sequences from the C-tails of aquaporin-2 or GluR1 recycled in a SAP97- and PKA-dependent manner. Non-PDZ β1-AR chimera derived from μ-opioid, dopamine 1, or GluR2 receptors promoted rapid recycling of chimeric β1-AR in a SAP97- and PKA-independent manner. Moreover, the nature of the residue at position -3 in the PDZ regulated whether the β1-AR was internalized alone or in complex with SAP97. These results indicate that divergent pathways were involved in trafficking the β1-AR and provide a roadmap for its trafficking via type I PDZs versus non-PDZs.
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Affiliation(s)
- Mohammed M Nooh
- From the Department of Pharmacology, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163 and
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15
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Huang P, Chiu YT, Chen C, Wang Y, Liu-Chen LY. A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells. J Pharmacol Toxicol Methods 2013; 68:340-5. [PMID: 23856011 PMCID: PMC3954113 DOI: 10.1016/j.vascn.2013.07.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2013] [Accepted: 07/04/2013] [Indexed: 12/21/2022]
Abstract
INTRODUCTION In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for the generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. METHODS We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into a Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. RESULTS KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of the time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. DISCUSSION tdT is an alternative to, or even a better tool than, GFPs for fusion to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to the reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity that the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance.
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Key Words
- 17,17′-(dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′-bimorphinan-3,4′,14,14′-tetrol
- DYKDDDDK epitope tag
- Dynorphin A (1-17)
- FLAG tag
- G protein-coupled receptor
- GFP
- GFPs
- GPCR
- H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH
- KOPR
- Live cell imaging
- MAPK
- Neurons
- RFP
- Red fluorescent protein
- U50,488H
- eGFP
- enhanced green fluorescent protein
- green fluorescent protein and its spectral variants
- kappa opioid receptor
- mKOPR
- mitogen-activated protein kinase
- mouse kappa opioid receptor
- norbinaltorphimine
- red fluorescent protein
- tdT
- tdTomato
- trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate salt
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Affiliation(s)
- Peng Huang
- Center for Substance Abuse Research and Department of Pharmacology,
Temple University School of Medicine, Philadelphia, Pennsylvania 19140
| | - Yi-Ting Chiu
- Center for Substance Abuse Research and Department of Pharmacology,
Temple University School of Medicine, Philadelphia, Pennsylvania 19140
| | - Chongguang Chen
- Center for Substance Abuse Research and Department of Pharmacology,
Temple University School of Medicine, Philadelphia, Pennsylvania 19140
| | - Yujun Wang
- Center for Substance Abuse Research and Department of Pharmacology,
Temple University School of Medicine, Philadelphia, Pennsylvania 19140
| | - Lee-Yuan Liu-Chen
- Center for Substance Abuse Research and Department of Pharmacology,
Temple University School of Medicine, Philadelphia, Pennsylvania 19140
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Abstract
In brain, properly balanced synaptic excitation and inhibition is critically important for network stability and efficient information processing. Here, we show that retinoic acid (RA), a synaptic signaling molecule whose synthesis is activated by reduced neural activity, induces rapid internalization of synaptic GABAA receptors in mouse hippocampal neurons, leading to significant reduction of inhibitory synaptic transmission. Similar to its action at excitatory synapses, action of RA at inhibitory synapses requires protein translation and is mediated by a nontranscriptional function of the RA-receptor RARα. Different from RA action at excitatory synapses, however, RA at inhibitory synapses causes a loss instead of the gain of a synaptic protein (i.e., GABAARs). Moreover, the removal of GABAARs from the synapses and the reduction of synaptic inhibition do not require the execution of RA's action at excitatory synapses (i.e., downscaling of synaptic inhibition is intact when upscaling of synaptic excitation is blocked). Thus, the action of RA at inhibitory and excitatory synapses diverges significantly after the step of RARα-mediated protein synthesis, and the regulations of GABAAR and AMPAR trafficking are independent processes. When both excitatory and inhibitory synapses are examined together in the same neuron, the synaptic excitation/inhibition ratio is significantly enhanced by RA. Importantly, RA-mediated downscaling of synaptic inhibition is completely absent in Fmr1 knock-out neurons. Thus, RA acts as a central organizer for coordinated homeostatic plasticity in both excitatory and inhibitory synapses, and impairment of this overall process alters the excitatory/inhibitory balance of a circuit and likely represents a major feature of fragile X-syndrome.
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17
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Reyes BAS, Chavkin C, Van Bockstaele EJ. Agonist-induced internalization of κ-opioid receptors in noradrenergic neurons of the rat locus coeruleus. J Chem Neuroanat 2010; 40:301-9. [PMID: 20884346 PMCID: PMC2991477 DOI: 10.1016/j.jchemneu.2010.09.003] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2010] [Revised: 09/20/2010] [Accepted: 09/20/2010] [Indexed: 12/11/2022]
Abstract
Kappa-opioid receptors (κOR) are positioned to modulate pre- and post-synaptic responses of norepinephrine-containing neurons in the rat locus coeruleus (LC). The ability of an acute systemic injection of a long acting κOR agonist, U50,488, to induce trafficking of κOR was assessed in the LC using immunogold-silver detection in male Sprague-Dawley rats. U50,488 administration shifted immunogold-silver labeling indicative of κOR from primarily plasmalemmal sites to intracellular sites when compared to vehicle-treated subjects. This translocation from the plasma membrane to the cytoplasmic compartment was prevented by pre-treatment with the κOR antagonist, norbinaltorphimine (norBNI). To determine whether agonist stimulation could induce adaptations in the expression of the noradrenergic synthesizing enzyme, dopamine beta hydroxylase (DβH), and κOR expression, Western blot analysis was used to compare expression levels of DβH and κOR following U50,488 administration. Expression levels for DβH and κOR were significantly increased following U50,488 administration when compared to controls. These data indicate that a systemic injection of a κOR agonist stimulates internalization of κORs in noradrenergic neurons and can impact κOR and DβH expression levels in this stress-sensitive brain region.
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MESH Headings
- 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/administration & dosage
- Animals
- Dopamine beta-Hydroxylase/biosynthesis
- Dopamine beta-Hydroxylase/genetics
- Endocytosis/drug effects
- Endocytosis/physiology
- Enkephalins/biosynthesis
- Enkephalins/genetics
- Locus Coeruleus/drug effects
- Locus Coeruleus/metabolism
- Locus Coeruleus/ultrastructure
- Male
- Microscopy, Immunoelectron
- Neurons/drug effects
- Neurons/metabolism
- Neurons/ultrastructure
- Norepinephrine/physiology
- Protein Precursors/biosynthesis
- Protein Precursors/genetics
- Rats
- Rats, Sprague-Dawley
- Receptors, Opioid, kappa/agonists
- Receptors, Opioid, kappa/metabolism
- Receptors, Opioid, kappa/ultrastructure
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Affiliation(s)
- B A S Reyes
- Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University, 900 Walnut Street, Philadelphia, PA 19107, USA.
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18
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Solinski HJ, Boekhoff I, Bouvier M, Gudermann T, Breit A. Sensory neuron-specific MAS-related gene-X1 receptors resist agonist-promoted endocytosis. Mol Pharmacol 2010; 78:249-59. [PMID: 20424127 DOI: 10.1124/mol.110.063867] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Human sensory neuron-specific mas-related gene X1 receptors (hMrgX1s) belong to the superfamily of G protein-coupled receptors (GPCRs), bind cleavage products of pro-enkephalin with high affinity, and have been suggested to participate in pain sensation. Murine or rat MrgC receptors exhibit high similarities with hMrgX1 in terms of expression pattern, sequence homology, and binding profile. Therefore, rodents have been used as an in vivo model to explore the physiological functions and pharmacodynamics of the hMrgX1. Agonist-promoted receptor endocytosis significantly affects the pharmacodynamics of a GPCR but is not yet investigated for hMrgX1. Therefore, we analyzed the effects of prolonged agonist exposure on cell surface protein levels of hMrgX1 and murine or rat MrgC in human embryonic kidney 293, Cos, F11, and ND-C cells. We observed that hMrgX1 are resistant and both MrgC are prone to agonist-promoted receptor endocytosis. In Cos cells, coexpression of beta-arrestins strongly enhanced endocytosis of murine MrgC but did not alter cell surface expression of hMrgX1 receptors. These data define the hMrgX1 as one of the few members within the superfamily of GPCRs whose signaling is not regulated by agonist-promoted endocytosis and reveal species-specific differences in the regulation of Mrg receptor signaling. Given the importance of receptor endocytosis for the pharmacodynamics of a given ligand, our results may have a strong impact on the development of future drugs that suppose to control pain in humans but were tested in rodents.
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Affiliation(s)
- Hans Jürgen Solinski
- Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München, Germany
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19
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von Zastrow M. Regulation of opioid receptors by endocytic membrane traffic: mechanisms and translational implications. Drug Alcohol Depend 2010; 108:166-71. [PMID: 20338697 PMCID: PMC3417350 DOI: 10.1016/j.drugalcdep.2010.02.014] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2009] [Revised: 02/17/2010] [Accepted: 02/19/2010] [Indexed: 11/27/2022]
Abstract
Opioid neuropeptide receptors mediate diverse physiological functions and are important targets for both therapeutic and abused drugs. Opioid receptors are highly regulated in intact cells, and there is reason to believe that this regulation controls the clinical effects of opioid drugs. The present review will discuss some of this evidence, focusing specifically on the regulation of opioid receptors by endocytic membrane trafficking mechanisms. First, some basic principles of regulated endocytosis will be reviewed, and the principle of 'molecular sorting' as a means to determine the functional consequences of endocytosis will be introduced, Most of this information has been derived from studies of simplified cell models. Second, present knowledge about the operation of these mechanisms in physiologically relevant CNS neurons will be discussed, focusing on studies of neurons cultured from rodent brain. Third, recent insight into the effects of endocytic trafficking on opioid regulation in vivo will be considered, focusing on results from studies of transgenic mouse models. Much remains to be learned at these pre-clinical levels, and effects of endocytosis on opioid actions in humans remain completely unexplored. Two particular insights, which have emerged from pre-clinical studies, will be proposed for translational consideration.
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Affiliation(s)
- Mark von Zastrow
- Department of Psychiatry, N212E Genentech Hall, UCSF Mission Bay Campus, San Francisco, CA 94158-2140, USA.
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20
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Marley A, von Zastrow M. Dysbindin promotes the post-endocytic sorting of G protein-coupled receptors to lysosomes. PLoS One 2010; 5:e9325. [PMID: 20174469 PMCID: PMC2824829 DOI: 10.1371/journal.pone.0009325] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2009] [Accepted: 02/01/2010] [Indexed: 01/27/2023] Open
Abstract
Background Dysbindin, a cytoplasmic protein long known to function in the biogenesis of specialized lysosome-related organelles (LROs), has been reported to reduce surface expression of D2 dopamine receptors in neurons. Dysbindin is broadly expressed, and dopamine receptors are members of the large family of G protein-coupled receptors (GPCRs) that function in diverse cell types. Thus we asked if dysbindin regulates receptor number in non-neural cells, and further investigated the cellular basis of this regulation. Methodology/Principal Findings We used RNA interference to deplete endogenous dysbindin in HEK293 and HeLa cells, then used immunochemical and biochemical methods to assess expression and endocytic trafficking of epitope-tagged GPCRs. Dysbindin knockdown up-regulated surface expression of D2 receptors compared to D1 receptors, as reported previously in neurons. This regulation was not mediated by a change in D2 receptor endocytosis. Instead, dysbindin knockdown specifically reduced the subsequent trafficking of internalized D2 receptors to lysosomes. This distinct post-endocytic sorting function explained the minimal effect of dysbindin depletion on D1 receptors, which recycle efficiently and traverse the lysosomal pathway to only a small degree. Moreover, dysbindin regulated the delta opioid receptor, a more distantly related GPCR that is also sorted to lysosomes after endocytosis. Dysbindin was not required for lysosomal trafficking of all signaling receptors, however, as its depletion did not detectably affect down-regulation of the EGF receptor tyrosine kinase. Dysbindin co-immunoprecipitated with GASP-1 (or GPRASP-1), a cytoplasmic protein shown previously to modulate lysosomal trafficking of D2 dopamine and delta opioid receptors by direct interaction, and with HRS that is a core component of the conserved ESCRT machinery mediating lysosome biogenesis and sorting. Conclusions/Significance These results identify a distinct, and potentially widespread function of dysbindin in promoting the sorting of specific GPCRs to lysosomes after endocytosis.
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Affiliation(s)
- Aaron Marley
- Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, United States of America
| | - Mark von Zastrow
- Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, United States of America
- * E-mail:
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21
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van Rijn RM, Whistler JL, Waldhoer M. Opioid-receptor-heteromer-specific trafficking and pharmacology. Curr Opin Pharmacol 2009; 10:73-9. [PMID: 19846340 DOI: 10.1016/j.coph.2009.09.007] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2009] [Revised: 09/16/2009] [Accepted: 09/17/2009] [Indexed: 12/22/2022]
Abstract
Homomerization and heteromerization of 7 transmembrane spanning (7TM)/G-protein-coupled receptors (GPCRs) have been an important field of study. Whereas initial studies were performed in artificial cell systems, recent publications are shifting the focus to the in vivo relevance of heteromerization. This is especially apparent for the field of opioid receptors. Drugs have been identified that selectively target opioid heteromers of the delta-opioid receptor with the kappa and the mu-opioid receptors that influence nociception and ethanol consumption, respectively. In addition, in several cases, the specific physiological response produced by the heteromer may be directly attributed to a difference in receptor trafficking properties of the heteromers compared with their homomeric counterparts. This review attempts to highlight some of the latest developments with regard to opioid receptor heteromer trafficking and pharmacology.
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Affiliation(s)
- Richard M van Rijn
- Ernest Gallo Clinic and Research Center, University of California San Francisco, Department of Neurology, Emeryville, CA 94608, USA
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22
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Lin H, Higgins P, Loh HH, Law PY, Liao D. Bidirectional effects of fentanyl on dendritic spines and AMPA receptors depend upon the internalization of mu opioid receptors. Neuropsychopharmacology 2009; 34:2097-111. [PMID: 19295508 PMCID: PMC2731771 DOI: 10.1038/npp.2009.34] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Fentanyl is a frequently used and abused opioid analgesic and can cause internalization of mu opioid receptors (MORs). Receptor internalization modulates the signaling pathways of opioid receptors. As changes in dendritic spines and synaptic AMPA receptors play important roles in addiction and memory loss, we investigated how fentanyl affects dendritic spines and synaptic AMPA receptors in cultured hippocampal neurons. Fentanyl at low concentrations (0.01 and 0.1 microM) caused the collapse of dendritic spines and decreased the number of AMPA receptor clusters. In contrast, fentanyl at high concentrations (1 and 10 microM) had opposite effects, inducing the emergence of new spines and increasing the number of AMPA receptor clusters. These dose-dependent bidirectional effects of fentanyl were blocked by a selective MOR antagonist CTOP at 5 microM. In neurons that had been transfected with HA-tagged or GFP-tagged MORs, fentanyl at high concentrations induced persistent and robust internalization of MORs, whereas fentanyl at lower concentrations induced little or transient receptor internalization. The blockade of receptor internalization with the expression of dominant-negative Dynamin I (the K44E mutant) reversed the effect of fentanyl at high concentrations, supporting a role of receptor internalization in modulating the dose-dependent effects of fentanyl. In contrast to morphine, the effects of fentanyl on dendritic spines are distinctively bidirectional and concentration dependent, probably due to its ability to induce robust internalization of MORs at high concentrations. The characterization of the effects of fentanyl on spines and AMPA receptors may help us understand the roles of MOR internalization in addiction and cognitive deficits.
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MESH Headings
- Analgesics, Opioid/administration & dosage
- Analgesics, Opioid/pharmacology
- Animals
- Cell Membrane/drug effects
- Cell Membrane/physiology
- Cells, Cultured
- Central Nervous System Agents
- Dendritic Spines/drug effects
- Dendritic Spines/physiology
- Dose-Response Relationship, Drug
- Dynamin I/genetics
- Dynamin I/metabolism
- Fentanyl/administration & dosage
- Fentanyl/pharmacology
- Hippocampus/drug effects
- Hippocampus/physiology
- Mutation, Missense
- Neurons/drug effects
- Neurons/physiology
- Protein Transport/drug effects
- Rats
- Receptors, AMPA/metabolism
- Receptors, Opioid, mu/antagonists & inhibitors
- Receptors, Opioid, mu/metabolism
- Somatostatin/analogs & derivatives
- Somatostatin/pharmacology
- Synapses/drug effects
- Synapses/physiology
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Affiliation(s)
- Hang Lin
- Department of Neuroscience, The University of Minnesota, 321 Church St S.E. Minneapolis, MN 55455
- Department of Neurology, Chengdu General Military Hospital, Chengdu City, 610083, China
| | - Paul Higgins
- Department of Neuroscience, The University of Minnesota, 321 Church St S.E. Minneapolis, MN 55455
| | - Horace H. Loh
- Department of Pharmacology, The University of Minnesota, 321 Church St S.E. Minneapolis, MN 55455
| | - Ping-Yee Law
- Department of Pharmacology, The University of Minnesota, 321 Church St S.E. Minneapolis, MN 55455
| | - Dezhi Liao
- Department of Neuroscience, The University of Minnesota, 321 Church St S.E. Minneapolis, MN 55455
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Functional characterization of human variants of the mu-opioid receptor gene. Proc Natl Acad Sci U S A 2009; 106:10811-6. [PMID: 19528663 DOI: 10.1073/pnas.0904509106] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Opioids and their receptors have an important role in analgesia and alcohol and substance use disorders (ASUD). We have identified several naturally occurring amino acid changing variants of the human mu-opioid receptor (MOR), and assessed the functional consequences of these previously undescribed variants in stably expressing cell lines. Several of these variants had altered trafficking and signaling properties. We found that an L85I variant showed significant internalization in response to morphine, in contrast to the WT MOR, which did not internalize in response to morphine. Also, when L85I and WT receptor were coexpressed, WT MOR internalized with the L85I MOR, suggesting that, in the heterozygous condition, the L85I phenotype would be dominant. This finding is potentially important, because receptor internalization has been associated with development of tolerance to opiate analgesics. In contrast, an R181C variant abolished both signaling and internalization in response to saturating doses of the hydrolysis-resistant enkephalin [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO). Coexpression of the R181C and WT receptor led to independent trafficking of the 2 receptors. S42T and C192F variants showed a rightward shift in potency of both morphine and DAMGO, whereas the S147C variant displayed a subtle leftward shift in morphine potency. These data suggest that these and other such variants may have clinical relevance to opioid responsiveness to both endogenous ligands and exogenous drugs, and could influence a broad range of phenotypes, including ASUD, pain responses, and the development of tolerance to morphine.
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Le Guyader L, Le Roux C, Mazères S, Gaspard-Iloughmane H, Gornitzka H, Millot C, Mingotaud C, Lopez A. Changes of the membrane lipid organization characterized by means of a new cholesterol-pyrene probe. Biophys J 2007; 93:4462-73. [PMID: 17766338 PMCID: PMC2098716 DOI: 10.1529/biophysj.107.112821] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
We synthesized 3β-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): KDOPC ≫ KPOPC ≫ KDMPC > KDMPC/15 mol % Chol > KDMPC/30 mol % Chol. This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I1–I5) and excimer (IE) emission bands. In model membranes, I1/I3 and IE/I3 ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse δ-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse δ-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.
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Raut A, Rao VR, Ratka A. Changes in opioid receptor proteins during mitochondrial impairment in differentiated SK-N-SH cells. Neurosci Lett 2007; 422:187-92. [PMID: 17611027 PMCID: PMC2112745 DOI: 10.1016/j.neulet.2007.06.015] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2007] [Revised: 05/31/2007] [Accepted: 06/11/2007] [Indexed: 11/20/2022]
Abstract
Aging and neurodegenerative diseases are associated with oxidative damage that may contribute to changes in neurosensory processing, including pain. The effects of neuronal oxidation on the opioid receptor system are poorly understood. Earlier, we have reported that 3-nitroproprionic acid (3-NPA)-induced oxidative stress and impairment of mitochondrial energy metabolism significantly reduced the function of mu but not delta opioid receptors [A. Raut, M. Iglewski, A. Ratka, Differential effects of impaired mitochondrial energy production on the function of mu and delta opioid receptors in neuronal SK-N-SH cells, Neurosci. Lett. 404 (2006) 242-246]. In the present study, we studied the effects of 3-NPA-induced oxidative stress on protein levels of the mu, delta, and kappa opioid receptors (MOR, DOR, and KOR, respectively). The opioid-responsive differentiated SK-N-SH neuronal cells were used as an in vitro model. Cells were exposed to 0, 5, 10, and 20mM of 3-NPA for 0, 1, 2, 12, and 24h. After the 3-NPA treatments, plasma membrane preparations were made and used for the Western blot assay. There was a significant reduction in the level of the MOR protein while levels of DOR and KOR proteins remained unaffected after exposure to 3-NPA. These findings demonstrate for the first time that there is a selective impairment of the MOR protein under conditions of mitochondrial oxidative damage at the neuronal level. The reduction in the level of the MOR protein may contribute to the impairment of MOR function under oxidative damage conditions shown in our previous study.
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Affiliation(s)
- Atul Raut
- Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, TX 76107, USA
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Liao D, Grigoriants OO, Wang W, Wiens K, Loh HH, Law PY. Distinct effects of individual opioids on the morphology of spines depend upon the internalization of mu opioid receptors. Mol Cell Neurosci 2007; 35:456-69. [PMID: 17513124 PMCID: PMC1931568 DOI: 10.1016/j.mcn.2007.04.007] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2006] [Revised: 04/06/2007] [Accepted: 04/19/2007] [Indexed: 12/28/2022] Open
Abstract
This study has examined the relationship between the effects of opioids on the internalization of mu opioid receptors (MORs) and the morphology of dendritic spines. Several opioids (morphine, etorphine, DAMGO or methadone) were applied to cultured hippocampal neurons. Live imaging and biochemical techniques were used to examine the dynamic changes in MOR internalization and spine morphology. This study reveals that MOR internalization can regulate opioid-induced morphological changes in dendritic spines: (1) Chronic treatment with morphine, which induced minimal receptor internalization, caused collapse of dendritic spines. In contrast, "internalizing" opioids such as DAMGO and etorphine induced the emergence of new spines. It reveals that opioid-induced changes in spines vary greatly depending on how the applied opioid agonist affects MOR internalization. (2) The blockade of receptor internalization by dominant negative mutant of dynamin, K44E, reversed the effects of DAMGO and etorphine. It indicates that receptor internalization is necessary for the distinct effects of DAMGO and etorphine on spines. (3) In neurons that were cultured from MOR knock-out mice and had been co-transfected with DsRed and MOR-GFP, morphine caused collapse of spines whereas DAMGO induced emergence of new spines, indicating that opioids can alter the structure of spines via postsynaptic MORs. (4) Methadone at a low concentration induced minimal internalization and had effects that were similar to morphine. At a high concentration, methadone induced robust internalization and had effects that are opposite to morphine. The concentration-dependent opioid-induced changes in dendritic spines might also contribute to the variation in the effects of individual opioids.
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Affiliation(s)
- Dezhi Liao
- Department of Neuroscience and Basic Research Center on Molecular and Cell Biology of Drug Addiction, The University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, USA.
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Yadav PN, Chaturvedi K, Howells RD. Inhibition of agonist-induced down-regulation of the delta-opioid receptor with a proteasome inhibitor attenuates opioid tolerance in human embryonic kidney 293 cells. J Pharmacol Exp Ther 2007; 320:1186-94. [PMID: 17159161 DOI: 10.1124/jpet.106.113621] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
This study was designed to test the hypothesis that inhibition of agonist-induced delta-receptor down-regulation would block the development of opioid tolerance in a cell-based model. A human embryonic kidney 293 cell line was established that expressed an epitope-tagged delta-opioid receptor (DOR). Treatment of DOR cells with Tyr-d-Ala-Gly-Phe-d-Leu-enkephalin (DADL) resulted in a time-dependent decrease in the B(max) of delta-opioid receptor binding sites and immunoreactive receptor protein. When cells were coincubated with the proteasome inhibitor N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (ZLLL) and DADL, the magnitude of the agonist-induced decrease in B(max) and immunoreactive receptor protein was reduced compared with DADL treatment alone. Acute treatment of DOR cells with DADL caused a 3-fold increase in the level of phosphorylated mitogen-activated protein (MAP) kinase. Prior exposure of DOR cells to DADL completely abrogated the agonist-induced activation of MAP kinase. When DOR cells were coincubated with DADL and ZLLL, the proteasome inhibitor prevented the loss of agonist activation of MAP kinase. Acute treatment of DOR cell membranes with DADL stimulated [(35)S]guanosine 5'-3-O-(thio-)triphosphate (GTPgammaS) binding. When DOR cells were preincubated with DADL, the agonist-induced increase in [(35)S]GTPgammaS binding was attenuated. Coincubation of ZLLL and agonist partially prevented the decreased responsiveness to agonist stimulation. The results of this study demonstrated that inhibition of agonist-induced down regulation with a proteasome inhibitor attenuated opioid tolerance in a cellular model, and suggest that coadministration of a proteasome inhibitor with chronic opioid agonist treatment may be useful for limiting opioid tolerance in vivo.
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Affiliation(s)
- Prem N Yadav
- Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, USA
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Huang P, Xu W, Yoon SI, Chen C, Chong PLG, Liu-Chen LY. Cholesterol reduction by methyl-beta-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells. Biochem Pharmacol 2007; 73:534-49. [PMID: 17141202 PMCID: PMC2583444 DOI: 10.1016/j.bcp.2006.10.032] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2006] [Revised: 10/30/2006] [Accepted: 10/31/2006] [Indexed: 11/30/2022]
Abstract
Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na(2)CO(3) buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted approximately 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-beta-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [(35)S]GTPgammaS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alphai) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alphai/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.
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Affiliation(s)
- Peng Huang
- Department of Pharmacology, Center for Substance Abuse Research, Temple University School of Medicine, 3420 N Broad Street, Philadelphia, PA 19140, United States
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Marie N, Aguila B, Allouche S. Tracking the opioid receptors on the way of desensitization. Cell Signal 2006; 18:1815-33. [PMID: 16750901 DOI: 10.1016/j.cellsig.2006.03.015] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2006] [Accepted: 03/21/2006] [Indexed: 11/24/2022]
Abstract
Opioid receptors belong to the super family of G-protein coupled receptors (GPCRs) and are the targets of numerous opioid analgesic drugs. Prolonged use of these drugs results in a reduction of their effectiveness in pain relief also called tolerance, a phenomenon well known by physicians. Opioid receptor desensitization is thought to play a major role in tolerance and a lot of work has been dedicated to elucidate the molecular basis of desensitization. As described for most of GPCRs, opioid receptor desensitization involves their phosphorylation by kinases and their uncoupling from G-proteins realized by arrestins. More recently, opioid receptor trafficking was shown to contribute to desensitization. In this review, our knowledge on the molecular mechanisms of desensitization and recent progress on the role of opioid receptor internalization, recycling or degradation in desensitization will be reported. A better understanding of these regulatory mechanisms would be helpful to develop new analgesic drugs or new strategies for pain treatment by limiting opioid receptor desensitization and tolerance.
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Affiliation(s)
- Nicolas Marie
- Neuropsychopharmacologie des addictions, CNRS 7157, INSERM U705, Université Paris V, France
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Raut A, Iglewski M, Ratka A. Differential effects of impaired mitochondrial energy production on the function of mu and delta opioid receptors in neuronal SK-N-SH cells. Neurosci Lett 2006; 404:242-6. [PMID: 16808998 DOI: 10.1016/j.neulet.2006.05.055] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2006] [Revised: 05/17/2006] [Accepted: 05/31/2006] [Indexed: 10/24/2022]
Abstract
Oxidative stress contributes to changes in neurosensory processing, including pain, that occur during aging and neurodegeneration. The effects of neuronal oxidation on the opioid system are poorly understood. In this in vitro study, oxidative stress was induced by 3-nitroproprionic acid (3-NPA) in opioid-responsive differentiated SK-N-SH cells. Changes in the inhibitory effects of opioid receptor agonists on intracellular cAMP were used as a marker of the function of mu and delta opioid receptors (MOR and DOR, respectively). Cells were treated with morphine and selective MOR and DOR agonists and antagonists to characterize the function of each receptor subtype. Cyclic AMP (cAMP) was measured by enzyme immunoassay. Levels of reactive oxygen species (ROS) were assessed using the 2',7'-dichlorofluorescin diacetate assay. Exposure of cells to 3-NPA resulted in an increase in ROS. After 3-NPA exposure, there was a significant attenuation of the inhibitory effect of morphine and DAMGO but not of DPDPE on cAMP. In cells pretreated with CTOP, 3-NPA did not change the inhibitory effect on cAMP. These findings demonstrate for the first time that under conditions of mitochondrial damage, the function of MOR is significantly decreased, while the function of DOR does not change, suggesting that the effect of 3-NPA on opioid receptors is subtype-specific.
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MESH Headings
- Cell Line, Tumor
- Energy Metabolism/physiology
- Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology
- Enkephalin, D-Penicillamine (2,5)-/pharmacology
- Humans
- Mitochondria/metabolism
- Neuroblastoma
- Neurons/physiology
- Nitro Compounds/pharmacology
- Propionates/pharmacology
- Reactive Oxygen Species/metabolism
- Receptors, Opioid, delta/drug effects
- Receptors, Opioid, delta/physiology
- Receptors, Opioid, mu/drug effects
- Receptors, Opioid, mu/physiology
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Affiliation(s)
- Atul Raut
- Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, 76107, USA
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Ramoino P, Gallus L, Beltrame F, Diaspro A, Fato M, Rubini P, Stigliani S, Bonanno G, Usai C. Endocytosis of GABAB receptors modulates membrane excitability in the single-celled organism Paramecium. J Cell Sci 2006; 119:2056-64. [PMID: 16638809 DOI: 10.1242/jcs.02931] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.
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Affiliation(s)
- Paola Ramoino
- Department for the Study of the Territory and its Resources (DIP.TE.RIS.), University of Genoa, Corso Europa 26, 16132 Genova, Italy.
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Zhao H, Loh HH, Law PY. Adenylyl cyclase superactivation induced by long-term treatment with opioid agonist is dependent on receptor localized within lipid rafts and is independent of receptor internalization. Mol Pharmacol 2006; 69:1421-32. [PMID: 16415176 DOI: 10.1124/mol.105.020024] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Long-term opioid agonist treatment results in adenylyl cyclase superactivation. A recent "RAVE" theory implicates a direct correlation between the ability of agonist to induce receptor internalization and the magnitude of adenylyl cyclase superactivation. We decided to test such a theory by examining the adenylyl cyclase superactivation after long-term activation of mu-opioid receptor (MOR) in an EcR293 cell model. We examined the magnitudes of adenylyl cyclase superactivation in the presence of naloxone after long-term treatment with morphine, etorphine, and methadone, three agonists reported to have differential activities in promoting MOR internalization. It can be shown that the magnitudes of adenylyl cyclase superactivation after treating with these three agonists, although different, were dependent on MOR density. Blunting MOR internalization with the dominant-negative mutant of dynamin, K44E, did not alter the magnitude of either morphine- or etorphine-induced adenylyl cyclase superactivation. In the presence of diprenorphine, the magnitude of adenylyl cyclase superactivation after etorphine treatment was identical to that observed with morphine. It could be demonstrated further that adenylyl cyclase superactivation is dependent on the cell surface-located MOR. Sucrose gradient fractionation demonstrated the colocalization of MOR and adenylyl cyclase V/VI with caveolin-1, a marker for lipid rafts. After long-term agonist treatment, the majority of MOR remained at the lipid rafts. Methyl-beta-cyclodextrin (MbetaCD) completely blunted the adenylyl cyclase superactivation and agonist-induced receptor internalization. These MbetaCD actions were reversed by incubating the cells with cholesterol. Thus, the adenylyl cyclase superactivation is not dependent on agonist-induced receptor internalization. Rather, the location of MOR at lipid rafts is an absolute requirement for the observed adenylyl cyclase superactivation.
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Affiliation(s)
- Hui Zhao
- Department of Pharmacology, 6-120 Jackson Hall, Medical School, University of Minnesota, 321 Church St. S.E., Minneapolis, MN 55455-0217, USA
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Haberstock-Debic H, Kim KA, Yu YJ, von Zastrow M. Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J Neurosci 2006; 25:7847-57. [PMID: 16120787 PMCID: PMC6725258 DOI: 10.1523/jneurosci.5045-04.2005] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Morphine activates mu-opioid receptors (MORs) without promoting their rapid endocytosis in a number of cell types. A previous study suggested that morphine can drive rapid redistribution of MORs in the nucleus accumbens, but it was not possible in this in vivo study to identify a specific membrane trafficking pathway affected by morphine, to exclude possible indirect actions of morphine via opiate-regulated neural circuitry, or to define the mechanism of this morphine-dependent regulation. In the present study, we addressed these questions using dissociated primary cultures of rat striatal neurons as a model system. Morphine promoted a rapid redistribution of both endogenous and recombinant MORs within 30 min after drug addition to the culture medium. This effect was mediated by rapid endocytosis and occurred in a cell-autonomous manner, as indicated by its detection in cells plated at low density and in cultures in which depolarization was blocked by tetrodotoxin. Morphine-induced endocytosis of MORs was quantitatively similar to that induced by the enkephalin analog D-Ala2-N-Me-Phe4-Glycol5-enkephalin, and endocytosis induced by both ligands was inhibited by a dominant-negative mutant version of arrestin-3 (beta-arrestin-2). These results extend previous in vivo results and indicate that morphine is indeed capable of driving rapid endocytosis of mu-opioid receptors in an important subset of opiate-responsive CNS neurons. They also suggest a cellular mechanism by which beta-arrestins may modulate the physiological effects of morphine in vivo.
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Narita M, Suzuki M, Narita M, Niikura K, Nakamura A, Miyatake M, Yajima Y, Suzuki T. mu-Opioid receptor internalization-dependent and -independent mechanisms of the development of tolerance to mu-opioid receptor agonists: Comparison between etorphine and morphine. Neuroscience 2006; 138:609-19. [PMID: 16417975 DOI: 10.1016/j.neuroscience.2005.11.046] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2005] [Revised: 11/10/2005] [Accepted: 11/19/2005] [Indexed: 12/30/2022]
Abstract
A growing body of evidences suggests that receptor desensitization is implicated in the development of tolerance to opioids, which is generally regulated by protein kinases and receptor trafficking proteins. In the present study, we demonstrated that repeated s.c. treatment with etorphine, but not morphine, produced a significant increase in protein levels of G protein-coupled receptor kinase 2, dynamin II, beta-arrestin 2 and phosphorylated-conventional protein kinase C in membranes of the mouse spinal cord, suggesting that the etorphine-induced mu-opioid receptor desensitization may result from G protein-coupled receptor kinase 2/dynaminII/beta-arrestin2-dependent phosphorylation of mu-opioid receptors. Unlike etorphine, morphine failed to change the levels of these trafficking proteins. Furthermore, we found that the level of glial fibrillary acidic protein in the mouse spinal cord was clearly increased by chronic in vivo and in vitro treatment with morphine, whereas no such effect was noted by etorphine. In the behavioral study, intraperitoneal pretreatment with the glial-modulating agent propentofylline suppressed the development of tolerance to morphine-induced antinociception. In addition, intrathecal injection of astrocytes and astrocyte-conditioned medium mixture, which were obtained from cultured astrocytes of the newborn mouse spinal cord, aggravated the development of tolerance to morphine. In contrast, these agents failed to affect the development of tolerance induced by etorphine. These findings provide direct evidence for the distinct mechanisms between etorphine and morphine on the development of tolerance to spinal antinociception. These findings raise the possibility that the increased astroglia response produced by chronic morphine could be associated with the lack of mu-opioid receptor internalization.
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Affiliation(s)
- M Narita
- Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
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King T, Ossipov MH, Vanderah TW, Porreca F, Lai J. Is paradoxical pain induced by sustained opioid exposure an underlying mechanism of opioid antinociceptive tolerance? Neurosignals 2006; 14:194-205. [PMID: 16215302 DOI: 10.1159/000087658] [Citation(s) in RCA: 139] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2005] [Indexed: 12/29/2022] Open
Abstract
Opiates are the primary treatment for pain management in cancer patients reporting moderate to severe pain, and are being increasingly used for non-cancer chronic pain. However, prolonged administration of opiates is associated with significant problems including the development of antinociceptive tolerance, wherein higher doses of the drug are required over time to elicit the same amount of analgesia. High doses of opiates result in serious side effects such as constipation, nausea, vomiting, dizziness, somnolence, and impairment of mental alertness. In addition, sustained exposure to morphine has been shown to result in paradoxical pain in regions unaffected by the initial pain complaint, and which may also result in dose escalation, i.e. 'analgesic tolerance'. A concept that has been gaining considerable experimental validation is that prolonged use of opioids elicits paradoxical, abnormal pain. This enhanced pain state requires additional opioids to maintain a constant level of antinociception, and consequently may be interpreted as antinociceptive tolerance. Many substances have been shown to block or reverse antinociceptive tolerance. A non-inclusive list of examples of substances reported to block or reverse opioid antinociceptive tolerance include: substance P receptor (NK-1) antagonists, calcitonin gene-related peptide (CGRP) receptor antagonists, nitric oxide (NO) synthase inhibitors, calcium channel blockers, cyclooxygenase (COX) inhibitors, protein kinase C inhibitors, competitive and non-competitive antagonists of the NMDA (N-methyl-D-aspartate) receptor, AMPA (alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid) antagonists, anti-dynorphin antiserum, and cholecystokinin (CCK) receptor antagonists. Without exception, these substances are also antagonists of pain-enhancing agents. Prolonged opiate administration indeed induces upregulation of substance P (SP) and calcitonin gene-related peptide (CGRP) within sensory fibers in vivo, and this is accompanied by an enhanced release of excitatory neurotransmitters and neuropeptides from primary afferent fibers upon stimulation. The enhanced evoked release of neuropeptides is correlated with the onset of abnormal pain states and opioid antinociceptive tolerance. Importantly, the descending pain modulatory pathway from the brainstem rostral ventromedial medulla (RVM) via the dorsolateral funiculus (DLF) is critical for maintaining the changes observed in the spinal cord, abnormal pain states and antinociceptive tolerance, because animals with lesion of the DLF did not show enhanced evoked neuropeptide release, or develop abnormal pain or antinociceptive tolerance upon sustained exposure to opiates. Microinjection of either lidocaine or a CCK antagonist into the RVM blocked both thermal and touch hypersensitivity as well as antinociceptive tolerance. Thus, prolonged opioid exposure enhances a descending pain facilitatory pathway from the RVM that is mediated at least in part by CCK activity and is essential for the maintenance of antinociceptive tolerance.
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Affiliation(s)
- Tamara King
- Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA
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36
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Renigunta V, Yuan H, Zuzarte M, Rinné S, Koch A, Wischmeyer E, Schlichthörl G, Gao Y, Karschin A, Jacob R, Schwappach B, Daut J, Preisig-Müller R. The Retention Factor p11 Confers an Endoplasmic Reticulum-Localization Signal to the Potassium Channel TASK-1. Traffic 2005; 7:168-81. [PMID: 16420525 DOI: 10.1111/j.1600-0854.2005.00375.x] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.
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Affiliation(s)
- Vijay Renigunta
- Institute of Physiology, Marburg University, Deutschhausstr. 2, 35037 Marburg, Germany
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37
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Ryman-Rasmussen JP, Nichols DE, Mailman RB. Differential activation of adenylate cyclase and receptor internalization by novel dopamine D1 receptor agonists. Mol Pharmacol 2005; 68:1039-48. [PMID: 15985612 DOI: 10.1124/mol.105.012153] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Structurally dissimilar dopamine D(1) receptor agonists were compared with dopamine in their ability to activate adenylate cyclase and to internalize hemagglutinin-tagged human D(1) receptors in a stably transfected human embryonic kidney cell line. Thirteen dopamine D(1) receptor agonists were selected rationally from three different structural classes: rigid fused ring compounds [dihydrexidine, dinapsoline, dinoxyline, apomorphine, and (5aR,11bS)-4,5,5a,6,7,11b-hexahydro-2-propyl-3-thia-5-azacyclopent-1-ena[c]-phenanthrene-9,10-diol (A86929)]; isochromans [(1R,3S)-3-(1'adamantyl)-1-aminomethyl-3,4-dihydo-5,6-dihydroxy-1H-2-benzopyran (A77636) and (1R,3S)-3-phenyl-1-aminomethyl-3,4-dihydo-5,6-dihydroxy-1H-2-benzopyran (A68930)]; and benzazepines [7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF38393), (+/-)-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF77434), 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), 3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-]H-3-benzazepine (SKF83959), R(+)-6-chloro-7,8,-dihydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82957), and R(+)-6-chloro-7,8,-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297)]. The working hypothesis was that some agonists have differential effects on adenylate cyclase versus receptor internalization that could be correlated to the structural class of the agonist. First, the affinity for the hemagglutinin-hD(1) receptor and the intrinsic activity and potency of adenylate cyclase activation were determined for each compound. The internalization time course and internalization efficacy were then determined for each agonist. It was surprising that internalization efficacy was found to be independent of either agonist structural class or affinity. Only agonists that had both high adenylate cyclase functional potency and high intrinsic activity caused internalization. In addition, four agonists from two structural classes were identified that were capable of fully activating adenylate cyclase without eliciting an internalization response. This study provides the first extensive characterization of D(1) receptor internalization in response to structurally diverse agonists and, at least for the D(1) receptor, shows that functional selectivity is not predictable by simple structural examination. These data are consistent with the hypothesis that functional selectivity reflects subtle ligand-induced conformational changes as opposed to simple agonist trafficking among discrete receptor active states.
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Affiliation(s)
- Jessica P Ryman-Rasmussen
- Department of Psychiatry, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA
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38
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Karaji AG, Khansari N, Ansary B, Dehpour AR. Detection of opioid receptors on murine lymphocytes by indirect immunofluorescence: mature normal and tumor bearing mice lymphocytes. Int Immunopharmacol 2005; 5:1019-27. [PMID: 15829417 DOI: 10.1016/j.intimp.2005.01.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2004] [Revised: 12/29/2004] [Accepted: 01/28/2005] [Indexed: 10/25/2022]
Abstract
Opioid peptides modulate immune responses via ligation to classical opioid receptors (mu, delta and kappa), expressed on immune cells, or in an indirect fashion via the central nervous system. The combination of immunofluorescent technique and flow cytometry has proven to be sensitive methods for detection of opioid receptors on leukocytes. In the current study a fluorescein isothiocyanate-conjugated naltrexone (FITC-NTX) derivative in the absence or presence of naltrexone, as a competitor, was used to detect opioid receptors on thymocytes and then on splenocytes of normal and tumor bearing Balb/c mice. Tumor bearing mice were made by intraperitoneal injection of fibrosarcoma cell line. In a two weeks interval, tumor grew and then mice splenocytes were harvested. Cells were incubated with FITC-NTX alone (direct fluorescence), or FITC-NTX followed by biotin-conjugated anti-fluorescein IgG and extravidin-R-phycoerythrin (indirect immunofluorescence). Using flow cytometry we found that, with direct fluorescence staining there is only nonspecific cell staining. In contrast, indirect staining of cells demonstrated labeling of opioid receptors. Thymocytes displayed 37.5+/-7% specific labeling by current staining procedure. However, this specific staining was 17.2+/-4% and 7.5+/-2% in splenocytes of normal and tumor bearing mice, respectively. Taken together, these results showed that, direct fluorescence staining failed to stain opioid receptors expressed on lymphocytes. These receptors can only be detected by a biotin-streptavidin amplification procedure. We also found that the level of opioid receptors on mature lymphocytes is less than that of immature ones and are even lesser in the tumor bearing mice lymphocytes.
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Affiliation(s)
- Ali Gorgin Karaji
- Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
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39
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Waldhoer M, Fong J, Jones RM, Lunzer MM, Sharma SK, Kostenis E, Portoghese PS, Whistler JL. A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proc Natl Acad Sci U S A 2005; 102:9050-5. [PMID: 15932946 PMCID: PMC1157030 DOI: 10.1073/pnas.0501112102] [Citation(s) in RCA: 259] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
There has been much speculation regarding the functional relevance of G protein-coupled receptor heterodimers, primarily because demonstrating their existence in vivo has proven to be a considerable challenge. Here we show that the opioid agonist ligand 6'-guanidinonaltrindole (6'-GNTI) has the unique property of selectively activating only opioid receptor heterodimers but not homomers. Importantly, 6'-GNTI is an analgesic, thereby demonstrating that opioid receptor heterodimers are indeed functionally relevant in vivo. However, 6'-GNTI induces analgesia only when it is administered in the spinal cord but not in the brain, suggesting that the organization of heterodimers is tissue-specific. This study demonstrates a proof of concept for tissue-selective drug targeting based on G protein-coupled receptor heterodimerization. Importantly, targeting opioid heterodimers could provide an approach toward the design of analgesic drugs with reduced side effects.
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Affiliation(s)
- Maria Waldhoer
- Ernest Gallo Clinic and Research Center, University of California, San Francisco, CA 94608, USA
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40
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Allen JA, Yu JZ, Donati RJ, Rasenick MM. Beta-adrenergic receptor stimulation promotes G alpha s internalization through lipid rafts: a study in living cells. Mol Pharmacol 2005; 67:1493-504. [PMID: 15703379 DOI: 10.1124/mol.104.008342] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of G alpha s, we developed a functional G alpha s-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing G alpha s-GFP were treated with 10 microM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, G alpha s-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing G alpha s-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that G alpha s does not traffic through common endocytic pathways. Furthermore, G alpha s-GFP did not colocalize with internalized beta2-adrenergic receptors, suggesting that G alpha s and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated G alpha s-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased G alpha s protein in Triton X-100 -insoluble membrane fractions, suggesting that G alpha s moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of G alpha s-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated G alpha s moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of G alpha s plays a specific role in G protein-coupled receptor-mediated signaling and could enable G alpha s to traffic into the cellular interior to regulate effectors at multiple cellular sites.
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Affiliation(s)
- John A Allen
- Department of Physiology and Biophysics, University of Illinois at Chicago (UIC), 60612-7342, USA
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41
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Christoffers KH, Li H, Howells RD. Purification and mass spectrometric analysis of the delta opioid receptor. ACTA ACUST UNITED AC 2005; 136:54-64. [PMID: 15893587 DOI: 10.1016/j.molbrainres.2005.01.016] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2004] [Revised: 12/28/2004] [Accepted: 01/08/2005] [Indexed: 11/19/2022]
Abstract
A mouse delta opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B(max) of 10.5 pmol/mg protein. [3H]Bremazocine exhibited high affinity binding to the epitope-tagged delta opioid receptor with a K(D) of 1.4 nM. The agonists DADL, morphine, and DAMGO competitively inhibited bremazocine binding to the tagged delta receptor with K(I)'s of 0.9, 370, and 620 nM, respectively. Chronic treatment of cells expressing the epitope-tagged delta receptor with DADL resulted in downregulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The delta receptor was solubilized from HEK 293 cell membranes with n-dodecyl-beta-d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of Sephacryl S300 gel filtration chromatography, wheat germ agglutinin (WGA)-agarose chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and SDS/PAGE permitted purification of the receptor. The purified delta opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 65 kDa. MALDI-TOF mass spectrometry was used to identify and characterize peptides derived from the delta opioid receptor following in-gel digestion with trypsin, and precursor-derived ms/ms confirmed the identity of peptides derived from enzymatic digestion of the delta opioid receptor.
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MESH Headings
- Analgesics/pharmacokinetics
- Benzomorphans/pharmacokinetics
- Blotting, Western/methods
- Cell Line
- Chromatography, Affinity
- Chromatography, Gel/methods
- Humans
- Mass Spectrometry
- Models, Molecular
- Molecular Weight
- Radioligand Assay/methods
- Receptors, Opioid, delta/analysis
- Receptors, Opioid, delta/chemistry
- Receptors, Opioid, delta/drug effects
- Receptors, Opioid, delta/isolation & purification
- Solubility
- Transfection/methods
- Tritium/pharmacokinetics
- Trypsin/pharmacology
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Affiliation(s)
- Keith H Christoffers
- Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, USA
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42
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Varga EV, Navratilova E, Stropova D, Jambrosic J, Roeske WR, Yamamura HI. Agonist-specific regulation of the delta-opioid receptor. Life Sci 2005; 76:599-612. [PMID: 15567186 DOI: 10.1016/j.lfs.2004.07.020] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2004] [Accepted: 07/09/2004] [Indexed: 01/28/2023]
Abstract
Delta opioid receptor (DOR) agonists are attractive potential analgesics, since these compounds exhibit strong antinociceptive activity with relatively few side effects. In the past decade, several novel classes of delta-opioid agonists have been synthesized. Recent experimental data indicate that structurally distinct opioid agonists interact differently with the delta-opioid receptor. Consequently, individual agonist-bound DOR conformations may interact differently with intracellular proteins. In the present paper, after a brief review of the cellular processes that contribute to homologous desensitization of the DOR signaling, we shall focus on experimental data demonstrating that chemically different agonists differ in their ability to phosphorylate, internalize, and/or down-regulate the DOR. Homologous regulation of the opioid receptor signaling is thought to play an important role in the development of opioid tolerance. Therefore, agonist-specific differences in DOR regulation suggest that by further chemical modification, delta-selective opioid analgesics can be designed that exhibit a reduced propensity for analgesic tolerance.
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Affiliation(s)
- Eva V Varga
- Department of Pharmacology, and the Sarver Heart Center, The University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA
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43
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Wang Y, Tang K, Inan S, Siebert D, Holzgrabe U, Lee DYW, Huang P, Li JG, Cowan A, Liu-Chen LY. Comparison of pharmacological activities of three distinct kappa ligands (Salvinorin A, TRK-820 and 3FLB) on kappa opioid receptors in vitro and their antipruritic and antinociceptive activities in vivo. J Pharmacol Exp Ther 2005; 312:220-30. [PMID: 15383632 DOI: 10.1124/jpet.104.073668] [Citation(s) in RCA: 150] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Salvinorin A, TRK-820 (17-cyclopropylmethyl-3,14beta-dihydroxy-4,5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl) acrylamido]morphinan hydrochloride), and 3FLB (diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate) are structurally distinctly different from U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate], the prototypic selective kappa agonist. Here, we investigated their in vitro pharmacological activities on receptors expressed in Chinese hamster ovary cells and in vivo antiscratch and antinociceptive activities in mice. All three compounds showed high selectivity for the kappa opioid receptor (KOR) over the mu opioid receptor (MOR) and delta opioid receptor (DOR) and nociceptin or orphanin FQ receptors. In the guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, all three were full agonists on the KOR. The rank order of affinity and potency for the KOR was TRK-820 >> U50,488H approximately salvinorin A >> 3FLB. TRK-820 acted as a partial agonist on MOR and DOR, whereas salvinorin A and 3FLB showed no activities on these receptors. Salvinorin A, TRK-820, and 3FLB caused internalization of the human KOR in a dose-dependent manner. Interestingly, although salvinorin A and U50,488H had similar potencies in stimulating [(35)S]GTPgammaS binding, salvinorin A was about 40-fold less potent than U50,488H in promoting internalization. Following 4-h incubation, all three compounds induced down-regulation of the human KOR, with salvinorin A causing a lower extent of down-regulation. Although TRK-820 was potent and efficacious against compound 48/80-induced scratching, salvinorin A showed low and inconsistent effects, and 3FLB was inactive. In addition, salvinorin A and 3FLB were not active in the acetic acid abdominal constriction test. The discrepancy between in vitro and in vivo results may be due to in vivo metabolism of salvinorin A and 3FLB and possibly to their effects on other pharmacological targets.
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MESH Headings
- 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology
- Analgesics/pharmacology
- Animals
- Antipruritics/pharmacology
- Aza Compounds/pharmacology
- Binding, Competitive
- Diterpenes/pharmacology
- Diterpenes, Clerodane
- Down-Regulation/drug effects
- Endocytosis/drug effects
- Endocytosis/physiology
- Female
- Guanosine 5'-O-(3-Thiotriphosphate)/metabolism
- Humans
- Hydrocarbons, Fluorinated/pharmacology
- Male
- Mice
- Morphinans/pharmacology
- Receptors, Opioid/metabolism
- Receptors, Opioid, delta/metabolism
- Receptors, Opioid, kappa/drug effects
- Receptors, Opioid, kappa/metabolism
- Receptors, Opioid, mu/metabolism
- Spiro Compounds/pharmacology
- Sulfur Radioisotopes
- Nociceptin Receptor
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Affiliation(s)
- Yulin Wang
- Department of Pharmacology, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, PA 19140, USA
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44
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Cao TT, Brelot A, von Zastrow M. The composition of the beta-2 adrenergic receptor oligomer affects its membrane trafficking after ligand-induced endocytosis. Mol Pharmacol 2005; 67:288-97. [PMID: 15492118 DOI: 10.1124/mol.104.003608] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The beta-2 adrenergic receptor (B2AR) is well known to form oligomeric complexes in vivo, but the functional significance of this process is not fully understood. The present results identify an effect of oligomerization of the human B2AR on the membrane trafficking of receptors after agonist-induced endocytosis in stably transfected human embryonic kidney 293 cells. A sequence present in the cytoplasmic tail of the B2AR has been shown previously to be required for efficient recycling of internalized receptors. Mutation of this sequence was observed to inhibit recycling not only of the receptor containing the mutation but also of the coexpressed wild-type B2AR. Coexpression of recycling-defective mutant B2ARs also enhanced proteolytic degradation of the wild-type B2AR after agonist-induced endocytosis, consistent with trafficking of both receptors to lysosomes in an oligomeric complex. Coexpression of the delta opioid receptor (DOR) at similar levels produced a much smaller effect on endocytic trafficking of the B2AR, even though DOR traverses a similar membrane pathway as recycling-defective mutant B2ARs. Biochemical studies confirmed that B2AR/B2AR-ala homomeric complexes form more readily than DOR/B2AR heteromers in expression-matched cell clones and support the hypothesis that B2AR/B2AR-ala complexes are not disrupted by agonist. These results suggest that a significant fraction of B2ARs exists in oligomeric complexes after ligand-induced endocytosis and that the composition of the oligomeric complex influences the sorting of endocytosed receptors between functionally distinct recycling and degradative membrane pathways.
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Affiliation(s)
- Tracy T Cao
- Genentech Hall, Rm N212E, University of California-San Francisco, 600 16th Street, San Francisco, CA 94143-2140, USA
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45
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Gage RM, Matveeva EA, Whiteheart SW, von Zastrow M. Type I PDZ ligands are sufficient to promote rapid recycling of G Protein-coupled receptors independent of binding to N-ethylmaleimide-sensitive factor. J Biol Chem 2004; 280:3305-13. [PMID: 15548537 DOI: 10.1074/jbc.m406934200] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the beta2 adrenergic receptor (beta2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the beta2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the beta2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the beta1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the beta2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.
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Affiliation(s)
- Robert M Gage
- Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco California 94143, USA
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46
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Tegeder I, Geisslinger G. Opioids as modulators of cell death and survival--unraveling mechanisms and revealing new indications. Pharmacol Rev 2004; 56:351-69. [PMID: 15317908 DOI: 10.1124/pr.56.3.2] [Citation(s) in RCA: 151] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Opioids are powerful analgesics but also drugs of abuse. Because opioid addicts are susceptible to certain infections, opioids have been suspected to suppress the immune response. This was supported by the finding that various immune-competent cells express opioid receptors and undergo apoptosis when treated with opioid alkaloids. Recent evidence suggests that opioids may also effect neuronal survival and proliferation or migrating properties of tumor cells. A multitude of signaling pathways has been suggested to be involved in these extra-analgesic effects of opioids. Growth-promoting effects were found to be mediated through Akt and Erk signaling cascades. Death-promoting effects have been ascribed to inhibition of nuclear factor-kappaB, increase of Fas expression, p53 stabilization, cytokine and chemokine release, and activation of nitric oxide synthase, p38, and c-Jun-N-terminal kinase. Some of the observed effects were inhibited with opioid receptor antagonists or pertussis toxin; others were unaffected. It is still unclear whether these properties are mediated through typical opioid receptor activation and inhibitory G-protein-signaling. The present review tries to unravel controversial findings and provides a hypothesis that may help to integrate diverse results.
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Affiliation(s)
- Irmgard Tegeder
- Pharmazentrum Frankfurt, Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, Germany.
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47
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Abstract
G protein-coupled receptors (GPCRs) modulate diverse physiological and behavioral signaling pathways by virtue of changes in receptor activation and inactivation states. Functional changes in receptor properties include dynamic interactions with regulatory molecules and trafficking to various cellular compartments at various stages of the life cycle of a GPCR. This review focuses on trafficking of GPCRs to the cell surface, stabilization there, and agonist-regulated turnover. GPCR interactions with a variety of newly revealed partners also are reviewed with the intention of provoking further analysis of the relevance of these interactions in GPCR trafficking, signaling, or both. The disease consequences of mislocalization of GPCRs also are described.
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Affiliation(s)
- Christopher M Tan
- Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA
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48
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Gardner LA, Delos Santos NM, Matta SG, Whitt MA, Bahouth SW. Role of the Cyclic AMP-dependent Protein Kinase in Homologous Resensitization of the β1-Adrenergic Receptor. J Biol Chem 2004; 279:21135-43. [PMID: 14990580 DOI: 10.1074/jbc.m313652200] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
A fundamental question in biology is how the various motifs in G protein-coupled receptors participate in the divergent functions orchestrated by these molecules. Here we describe a fundamental role for a serine residue at position 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) in endocytic recycling of the agonist-internalized receptor. In receptor recycling experiments that were monitored by confocal microscopy, the agonist-internalized wild-type (WT) beta(1)-AR recycled with a t(0.5) of 14 +/- 3 min. Mutagenesis of Ser(312) to alanine (Ser(312) --> Ala beta(1)-AR) or to the phosphoserine mimic aspartic acid (Ser(312) --> Asp beta(1)-AR) resulted in beta(1)-AR constructs that were pharmacologically indistinguishable from the WT beta(1)-AR. The internalized Ser(312) --> Asp beta(1)-AR recycled efficiently with a t(0.5) of 11 +/- 3 min, whereas the internalized Ser(312) --> Ala beta(1)-AR was not recycled or functionally resensitized through the endosomal pathway. Because this serine is a putative residue for phosphorylation by the cyclic AMP-dependent protein kinase (PKA), we examined the role of this kinase in recycling of the internalized beta(1)-AR. Inhibition of PKA biochemically or genetically using a dominant negative PKA construct blocked the recycling of the internalized WT beta(1)-AR. Phosphorylation studies revealed that the beta(1)-AR is partially phosphorylated by PKA and that phosphorylation of the beta(1)-AR by the catalytic subunit of PKA occurs exclusively at Ser(312). Our results identify a new signaling paradigm in which homologous activation of a kinase provides a reversible modification that shifts the itinerary of the internalized receptor toward recycling and resensitization. Therefore, PKA-mediated phosphorylation of G protein-coupled receptors might result in motif-dependent desensitization or resensitization.
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Affiliation(s)
- Lidia A Gardner
- Pharmacology and Molecular Sciences, University of Tennessee Health Sciences Center, 874 Union Avenue, Memphis, TN 38163, USA
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Popova JS, Rasenick MM. Clathrin-mediated endocytosis of m3 muscarinic receptors. Roles for Gbetagamma and tubulin. J Biol Chem 2004; 279:30410-8. [PMID: 15117940 DOI: 10.1074/jbc.m402871200] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Receptors as well as some G protein subunits internalize after agonist stimulation. It is not clear whether Galpha(q) or Gbetagamma undergo such regulated translocation. Recent studies demonstrate that m3 muscarinic receptor activation in SK-N-SH neuroblastoma cells causes recruitment of tubulin to the plasma membrane. This subsequently transactivates Galpha(q) and activates phospholipase Cbeta1. Interaction of tubulin-GDP with Gbetagamma at the offset of phospholipase Cbeta1 signaling appears involved in translocation of tubulin and Gbetagamma to vesicle-like structures in the cytosol (Popova, J. S., and Rasenick, M. M. (2003) J. Biol. Chem. 278, 34299-34308). The relationship of this internalization to the clathrin-mediated endocytosis of the activated m3 muscarinic receptors or Galpha(q) involvement in this process has not been clarified. To test this, SK-N-SH cells were treated with carbachol, and localization of Galpha(q), Gbetagamma, tubulin, clathrin, and m3 receptors were analyzed by both cellular imaging and biochemical techniques. Upon agonist stimulation both tubulin and clathrin translocated to the plasma membrane and co-localized with receptors, Galpha(q) and Gbetagamma. Fifteen minutes later receptors, Gbetagamma and tubulin, but not Galpha(q), internalized with the clathrin-coated vesicles. Coimmunoprecipitation of m3 receptors with Gbetagamma, tubulin, and clathrin from the cytosol of carbachol-treated cells was readily observed. These data suggested that Gbetagamma subunits might organize the formation of a multiprotein complex linking m3 receptors to tubulin since they interacted with both proteins. Such protein assemblies might explain the dynamin-dependent but beta-arrestin-independent endocytosis of m3 muscarinic receptors since tubulin interaction with dynamin might guide or insert the complex into clathrin-coated pits. This novel mechanism of internalization might prove important for other beta-arrestin-independent endocytic pathways. It also suggests cross-regulation between G protein-mediated signaling and the dynamics of the microtubule cytoskeleton.
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Affiliation(s)
- Juliana S Popova
- Department of Physiology, College of Medicine, University of Illinois, Chicago, Illinois 60612-7342, USA.
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Pei L, Lee FJS, Moszczynska A, Vukusic B, Liu F. Regulation of dopamine D1 receptor function by physical interaction with the NMDA receptors. J Neurosci 2004; 24:1149-58. [PMID: 14762133 PMCID: PMC6793575 DOI: 10.1523/jneurosci.3922-03.2004] [Citation(s) in RCA: 140] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Functional interactions between dopamine D1-like receptors and NMDA subtype glutamate receptors have been implicated in the maintenance of normal brain activity and neurological dysfunction. Although modulation of NMDA receptor functions by D1 receptor activation has been the subject of extensive investigation, little is known as to how the activation of NMDA receptors alters D1 function. Here we report that NMDA receptors regulate D1 receptor function via a direct protein-protein interaction mediated by the carboxyl tail regions of both receptors. In both cotransfected cells and cultured hippocampal neurons the activation of NMDA receptors increases the number of D1 receptors on the plasma membrane surface and enhances D1 receptor-mediated cAMP accumulation via a SNARE-dependent mechanism. Furthermore, overexpression of mini-genes encoding either NR1 or D1 carboxyl tail fragments disrupts the D1-NR1 direct protein-protein interaction and abolishes NMDA-induced changes in both D1 cell surface expression and D1-mediated cAMP accumulation. Our results demonstrate that the D1-NR1 physical interaction enables NMDA receptors to increase plasma membrane insertion of D1 receptors and provides a novel mechanism by which the activation of NMDA receptors upregulates D1 receptor function. Understanding the molecular mechanisms by which D1 and NMDA receptors functionally interact may provide insight toward elucidating the molecular neurobiological mechanisms involved in many neuropsychiatric illnesses, such as schizophrenia.
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Affiliation(s)
- Lin Pei
- Department of Neuroscience, Centre for Addiction and Mental Health, Clarke Division, Toronto, Ontario, M5T 1R8 Canada
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