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Tarasova E, Bogacheva P, Chernyshev K, Balezina O. Quantal size increase induced by the endocannabinoid 2-arachidonoylglycerol requires activation of CGRP receptors in mouse motor synapses. Synapse 2024; 78:e22281. [PMID: 37694983 DOI: 10.1002/syn.22281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2023] [Revised: 08/22/2023] [Accepted: 08/24/2023] [Indexed: 09/12/2023]
Abstract
In mouse motor synapses, the exogenous application of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG) increases acetylcholine (ACh) quantal size due to the activation of CB1 receptors and the stimulation of ACh vesicular uptake. In the present study, microelectrode recordings of miniature endplate potentials (MEPP) revealed that this effect of 2-AG is independent of brain-derived neurotrophic factor (BDNF) signaling but involves the activation of calcitonin gene-related peptide (CGRP) receptors along with CB1 receptors. Potentiation of MEPP amplitude in the presence of 2-AG was prevented by blockers of CGRP receptors and ryanodine receptors (RyR) and by inhibitors of phospholipase C (PLC) and Ca2+ /calmodulin-dependent protein kinase II (CaMKII). Therefore, we suggest a hypothetical chain of events, which starts from the activation of presynaptic CB1 receptors, involves PLC, RyR, and CaMKII, and results in CGRP release with the subsequent activation of presynaptic CGRP receptors. Activation of CGRP receptors is probably a part of a complex molecular cascade leading to the 2-AG-induced increase in ACh quantal size and MEPP amplitude. We propose that the same chain of events may also take place if 2-AG is endogenously produced in mouse motor synapses, because the increase in MEPP amplitude that follows after prolonged tetanic muscle contractions (30 Hz, 2 min) was prevented by the blocking of CB1 receptors. This work may help to unveil the previously unknown aspects of the functional interaction between ECs and peptide modulators aimed at the regulation of quantal size and synaptic transmission.
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Affiliation(s)
- Ekaterina Tarasova
- Department of Human and Animal Physiology, Biological Faculty, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Polina Bogacheva
- Department of Human and Animal Physiology, Biological Faculty, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Kirill Chernyshev
- Department of Human and Animal Physiology, Biological Faculty, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Olga Balezina
- Department of Human and Animal Physiology, Biological Faculty, Lomonosov Moscow State University, Moscow, Russian Federation
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2
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Ferraiolo M, Ponthot R, Atik H, Koener B, Hanson J, Hermans E. Receptor density influences the recruitment bias of aripiprazole and brexpiprazole at the dopamine D
2L
receptor. Fundam Clin Pharmacol 2022; 36:976-984. [DOI: 10.1111/fcp.12812] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2022] [Revised: 05/26/2022] [Accepted: 06/27/2022] [Indexed: 11/29/2022]
Affiliation(s)
- Mattia Ferraiolo
- Neuropharmacology Laboratory Institute of Neurosciences – UCLouvain Brussels Belgium
| | - Romane Ponthot
- Neuropharmacology Laboratory Institute of Neurosciences – UCLouvain Brussels Belgium
| | - Hicham Atik
- Neuropharmacology Laboratory Institute of Neurosciences – UCLouvain Brussels Belgium
| | - Beryl Koener
- Neuropharmacology Laboratory Institute of Neurosciences – UCLouvain Brussels Belgium
| | - Julien Hanson
- Laboratory of Molecular pharmacology GIGA‐Molecular Biology of Disease – ULiège Liège Belgium
- Laboratory of Medicinal Chemistry CIRM – ULiège Liège Belgium
| | - Emmanuel Hermans
- Neuropharmacology Laboratory Institute of Neurosciences – UCLouvain Brussels Belgium
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3
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Diversity of the Gβγ complexes defines spatial and temporal bias of GPCR signaling. Cell Syst 2021; 12:324-337.e5. [PMID: 33667409 DOI: 10.1016/j.cels.2021.02.001] [Citation(s) in RCA: 49] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Revised: 12/09/2020] [Accepted: 02/04/2021] [Indexed: 01/04/2023]
Abstract
The signal transduction by G-protein-coupled receptors (GPCRs) is mediated by heterotrimeric G proteins composed from one of the 16 Gα subunits and the inseparable Gβγ complex assembled from a repertoire of 5 Gβ and 12 Gγ subunits. However, the functional role of compositional diversity in Gβγ complexes has been elusive. Using optical biosensors, we examined the function of all Gβγ combinations in living cells and uncovered two major roles of Gβγ diversity. First, we demonstrate that the identity of Gβγ subunits greatly influences the kinetics and efficacy of GPCR responses at the plasma membrane. Second, we show that different Gβγ combinations are selectively dispatched from the plasma membrane to various cellular organelles on a timescale from milliseconds to minutes. We describe the mechanisms regulating these processes and document their implications for GPCR signaling via various Gα subunits, thereby illustrating a role for the compositional diversity of G protein heterotrimers.
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4
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Yang M, Zhang CY. G protein-coupled receptors as potential targets for nonalcoholic fatty liver disease treatment. World J Gastroenterol 2021; 27:677-691. [PMID: 33716447 PMCID: PMC7934005 DOI: 10.3748/wjg.v27.i8.677] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 12/24/2020] [Accepted: 01/21/2021] [Indexed: 02/06/2023] Open
Abstract
Nonalcoholic fatty liver disease (NAFLD) is a broad-spectrum disease, ranging from simple hepatic steatosis to nonalcoholic steatohepatitis, which can progress to cirrhosis and liver cancer. Abnormal hepatic lipid accumulation is the major manifestation of this disease, and lipotoxicity promotes NAFLD progression. In addition, intermediate metabolites such as succinate can stimulate the activation of hepatic stellate cells to produce extracellular matrix proteins, resulting in progression of NAFLD to fibrosis and even cirrhosis. G protein-coupled receptors (GPCRs) have been shown to play essential roles in metabolic disorders, such as NAFLD and obesity, through their function as receptors for bile acids and free fatty acids. In addition, GPCRs link gut microbiota-mediated connections in a variety of diseases, such as intestinal diseases, hepatic steatosis, diabetes, and cardiovascular diseases. The latest findings show that gut microbiota-derived acetate contributes to liver lipogenesis by converting dietary fructose into hepatic acetyl-CoA and fatty acids. GPCR agonists, including peptides and natural products like docosahexaenoic acid, have been applied to investigate their role in liver diseases. Therapies such as probiotics and GPCR agonists may be applied to modulate GPCR function to ameliorate liver metabolism syndrome. This review summarizes the current findings regarding the role of GPCRs in the development and progression of NAFLD and describes some preclinical and clinical studies of GPCR-mediated treatment. Overall, understanding GPCR-mediated signaling in liver disease may provide new therapeutic options for NAFLD.
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Affiliation(s)
- Ming Yang
- Department of Surgery, University of Missouri, Columbia, MO 65212, United States
| | - Chun-Ye Zhang
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65212, United States
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5
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Riquelme-Sandoval A, de Sá-Ferreira CO, Miyakoshi LM, Hedin-Pereira C. New Insights Into Peptide Cannabinoids: Structure, Biosynthesis and Signaling. Front Pharmacol 2020; 11:596572. [PMID: 33362550 PMCID: PMC7759141 DOI: 10.3389/fphar.2020.596572] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Accepted: 10/19/2020] [Indexed: 01/01/2023] Open
Abstract
Classically, the endocannabinoid system (ECS) consists of endogenous lipids, of which the best known are anandamide (AEA) and 2 arachidonoylglycerol (2-AG), their enzyme machinery for synthesis and degradation and their specific receptors, cannabinoid receptor one (CB1) and cannabinoid receptor two (CB2). However, endocannabinoids also bind to other groups of receptors. Furthermore, another group of lipids are considered to be endocannabinoids, such as the fatty acid ethanolamides, the fatty acid primary amides and the monoacylglycerol related molecules. Recently, it has been shown that the hemopressin peptide family, derived from α and β chains of hemoglobins, is a new family of cannabinoids. Some studies indicate that hemopressin peptides are expressed in the central nervous system and peripheral tissues and act as ligands of these receptors, thus suggesting that they play a physiological role. In this review, we examine new evidence on lipid endocannabinoids, cannabinoid receptors and the modulation of their signaling pathways. We focus our discussion on the current knowledge of the pharmacological effects, the biosynthesis of the peptide cannabinoids and the new insights on the activation and modulation of cannabinoid receptors by these peptides. The novel peptide compounds derived from hemoglobin chains and their non-classical activation of cannabinoid receptors are only starting to be uncovered. It will be exciting to follow the ensuing discoveries, not only in reference to what is already known of the classical lipid endocannabinoids revealing more complex aspects of endocannabinoid system, but also as to its possibilities as a future therapeutic tool.
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Affiliation(s)
- Agustín Riquelme-Sandoval
- Laboratory of Cellular Neuroanatomy, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Caio O de Sá-Ferreira
- Laboratory of Cellular Neuroanatomy, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.,Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Leo M Miyakoshi
- Laboratory of Cellular Neuroanatomy, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Cecilia Hedin-Pereira
- Laboratory of Cellular Neuroanatomy, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.,Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.,VPPCB-Fiocruz, Rio de Janeiro, Brazil.,National Institute of Science and Technology in Neuroimmunomodulation (INCT-NIM), Rio de Janeiro, Brazil
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6
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Lappano R, Mallet C, Rizzuti B, Grande F, Galli GR, Byrne C, Broutin I, Boudieu L, Eschalier A, Jacquot Y, Maggiolini M. The Peptide ERα17p Is a GPER Inverse Agonist that Exerts Antiproliferative Effects in Breast Cancer Cells. Cells 2019; 8:cells8060590. [PMID: 31207943 PMCID: PMC6627388 DOI: 10.3390/cells8060590] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Accepted: 06/13/2019] [Indexed: 12/18/2022] Open
Abstract
The inhibition of the G protein-coupled estrogen receptor (GPER) offers promising perspectives for the treatment of breast tumors. A peptide corresponding to part of the hinge region/AF2 domain of the human estrogen receptor α (ERα17p, residues 295–311) exerts anti-proliferative effects in various breast cancer cells including those used as triple negative breast cancer (TNBC) models. As preliminary investigations have evoked a role for the GPER in the mechanism of action of this peptide, we focused our studies on this protein using SkBr3 breast cancer cells, which are ideal for GPER evaluation. ERα17p inhibits cell growth by targeting membrane signaling. Identified as a GPER inverse agonist, it co-localizes with GPER and induces the proteasome-dependent downregulation of GPER. It also decreases the level of pEGFR (phosphorylation of epidermal growth factor receptor), pERK1/2 (phosphorylation of extracellular signal-regulated kinase), and c-fos. ERα17p is rapidly distributed in mice after intra-peritoneal injection and is found primarily in the mammary glands. The N-terminal PLMI motif, which presents analogies with the GPER antagonist PBX1, reproduces the effect of the whole ERα17p. Thus, this motif seems to direct the action of the entire peptide, as highlighted by docking and molecular dynamics studies. Consequently, the tetrapeptide PLMI, which can be claimed as the first peptidic GPER disruptor, could open new avenues for specific GPER modulators.
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Affiliation(s)
- Rosamaria Lappano
- Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy.
| | - Christophe Mallet
- NEURO-DOL Basics & Clinical Pharmacology of Pain, INSERM, CHU, Université Clermont Auvergne, F-63000 Clermont-Ferrand, France.
- ANALGESIA Institute, Université Clermont Auvergne, F-63000 Clermont-Ferrand, France.
| | - Bruno Rizzuti
- CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, 87036 Rende, Italy.
| | - Fedora Grande
- Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy.
| | - Giulia Raffaella Galli
- Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy.
| | - Cillian Byrne
- Laboratoire des Biomolécules (LBM), CNRS-UMR 7203, Sorbonne University, Ecole Normale Supérieure, 75252 Paris Cedex 05, France.
| | - Isabelle Broutin
- Cibles Thérapeutiques et Conception de Médicaments (CiTCoM), CNRS-UMR 8038, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 75270 Paris Cedex 06, France.
| | - Ludivine Boudieu
- NEURO-DOL Basics & Clinical Pharmacology of Pain, INSERM, CHU, Université Clermont Auvergne, F-63000 Clermont-Ferrand, France.
- ANALGESIA Institute, Université Clermont Auvergne, F-63000 Clermont-Ferrand, France.
| | - Alain Eschalier
- NEURO-DOL Basics & Clinical Pharmacology of Pain, INSERM, CHU, Université Clermont Auvergne, F-63000 Clermont-Ferrand, France.
- ANALGESIA Institute, Université Clermont Auvergne, F-63000 Clermont-Ferrand, France.
| | - Yves Jacquot
- Laboratoire des Biomolécules (LBM), CNRS-UMR 7203, Sorbonne University, Ecole Normale Supérieure, 75252 Paris Cedex 05, France.
| | - Marcello Maggiolini
- Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy.
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Kumar GA, Sarkar P, Jafurulla M, Singh SP, Srinivas G, Pande G, Chattopadhyay A. Exploring Endocytosis and Intracellular Trafficking of the Human Serotonin1A Receptor. Biochemistry 2019; 58:2628-2641. [DOI: 10.1021/acs.biochem.9b00033] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Affiliation(s)
- G. Aditya Kumar
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | - Parijat Sarkar
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | - Md. Jafurulla
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | - Shishu Pal Singh
- National Centre for Biological Sciences, UAS-GKVK Campus, Bellary Road, Bangalore 560 065, India
| | - Gunda Srinivas
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | - Gopal Pande
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
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Sarkar P, Chattopadhyay A. Exploring membrane organization at varying spatiotemporal resolutions utilizing fluorescence-based approaches: implications in membrane biology. Phys Chem Chem Phys 2019; 21:11554-11563. [DOI: 10.1039/c9cp02087j] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Representative experimental approaches based on dynamic fluorescence microscopy to analyze organization and dynamics of membrane lipids and proteins.
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Affiliation(s)
- Parijat Sarkar
- CSIR-Centre for Cellular and Molecular Biology
- Hyderabad 500 007
- India
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9
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Abstract
Delta opioid receptors (δORs) regulate a number of physiological functions, and agonists for this receptor are being pursued for the treatment of mood disorders, chronic pain, and migraine. A major challenge to the development of these compounds is that, like many G-protein coupled receptors (GPCRs), agonists at the δOR can induce very different signaling and receptor trafficking events. This concept, known as ligand-directed signaling, functional selectivity, or biased agonism, can result in different agonists producing highly distinct behavioral consequences. In this chapter, we highlight the in vitro and in vivo evidence for ligand-directed signaling and trafficking at the δOR. A number of biological implications of agonist-directed signaling at the δOR have been demonstrated. Importantly, ligand-specific effects can impact both acute behavioral effects of delta agonists, as well as the long-term adaptations induced by chronic drug treatment. A better understanding of the specific signaling cascades that regulate these differential behavioral effects would help to guide rational drug design, ultimately resulting in δOR agonists with fewer adverse effects.
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Affiliation(s)
- Ana Vicente-Sanchez
- Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA
| | - Amynah A Pradhan
- Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA.
- Department of Psychiatry, UIC, 1601 W Taylor St (MC 912), Chicago, IL, 60612, USA.
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10
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Abstract
In the past decade, automated microscopy has become an important tool for the drug discovery and development process. The establishment of imaging modalities as screening tools depended on technological breakthroughs in the domain of automated microscopy and automated image analysis. These types of assays are often referred to as high content screening or high content analysis (HCS/HCA). The driving force to adopt imaging for drug development is the quantity and quality of cellular information that can be collected and the enhanced physiological relevance of cellular screening compared to biochemical screening. Most imaging in drug development is performed on fixed cells as this allows uncoupling the preparation of the cells from the acquisition of the images. Live-cell imaging is technically challenging, but is very useful for many aspects of the drug development pipeline such as kinetic studies of compound mode of action or to analyze the motion of cellular components. Most vendors of HCS microscopy systems offer the option of environmental chambers and onboard pipetting on their platforms. This reflects the wish and desire of many customers to have the ability to perform live-cell assays on their HCS automated microscopes. This book chapter summarizes the challenges and advantages of live-cell imaging in drug discovery. Examples of applications are presented and the motivation to perform these assays in kinetic mode is discussed.
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Affiliation(s)
- Milan Esner
- High Throughput Technology Development Studio (HT-TDS), Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 3, 625 00, Brno, Czech Republic
| | - Felix Meyenhofer
- High Throughput Technology Development Studio (HT-TDS), Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany
- Département de Médecine, Faculté des Sciences, University of Fribourg, 1, Rte., Albert Gockel, Fribourg, 1700, Switzerland
| | - Marc Bickle
- High Throughput Technology Development Studio (HT-TDS), Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany.
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11
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Longitudinal trends and subgroup analysis in publication patterns for preclinical data of newly approved drugs. Naunyn Schmiedebergs Arch Pharmacol 2015; 389:201-9. [PMID: 26612506 DOI: 10.1007/s00210-015-1185-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Accepted: 10/09/2015] [Indexed: 10/22/2022]
Abstract
Having observed a large variation in the number and type of original preclinical publications for newly registered drugs, we have explored whether longitudinal trends and/or factors specific for certain drugs or their manufacturers may explain such variation. Our analysis is based on 1954 articles related to 170 newly approved drugs. The number of preclinical publications per compound declined from a median of 10.5 in 1991 to 3 in 2011. A similar trend was observed for the number of in vivo studies in general, but not in the subset of in vivo studies in animal models of disease. The percentage of compounds with studies using isolated human cells or cell lines almost doubled over time from 37 to 72%. Number of publications did not exhibit major differences between compounds intended for human versus veterinary use, therapeutic areas, small molecules versus biologicals, or innovator versus follow-up compounds; however, some companies may publish fewer studies per compound than others. However, there were qualitative differences in the types of models being used depending on the therapeutic area; specifically, compounds for use in oncology very often used isolated cells and cell lines, often from human origin. We conclude that the large variation in number and type of reported preclinical data is not easily explained. We propose that pharmaceutical companies should consistently provide a comprehensive documentation of the preclinical data they generate as part of their development programs in the public domain to enable a better understanding of the drugs they intend to market.
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12
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Matsumoto-Miyai K, Yoshizumi M, Kawatani M. Regulatory Effects of 5-Hydroxytryptamine Receptors on Voiding Function. Adv Ther 2015; 32 Suppl 1:3-15. [PMID: 26391372 DOI: 10.1007/s12325-015-0240-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2015] [Indexed: 10/23/2022]
Abstract
UNLABELLED A growing body of evidence suggests that 5-hydroxytryptamine (5-HT; serotonin) has both physiological and pathological functions in the lower urinary tract. A wide variety of 5-HT receptor subtypes are variably expressed in different organs, both peripheral and central. On urinary bladder smooth muscle, 5-HT1A, 5-HT2, 5-HT3, and 5-HT7 subtypes could function as postjunctional receptors. Postjunctional 5-HT2 receptors induce detrusor contraction of the bladder body. 5-HT1A is suggested to have a similar effect to 5-HT2, while 5-HT3 might suppress detrusor contraction evoked by direct muscle stimulation. Postjunctional 5-HT7 is reported to induce relaxation of the bladder neck, which might be required for efficient voiding. 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes also could act as prejunctional receptors in autonomic excitatory nerve terminals. 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes facilitate the neurogenic contraction of the detrusor by enhancing cholinergic or purinergic transmission, whereas 5-HT1A receptors might inhibit the release of acetylcholine in the detrusor. Furthermore, 5-HT1D could be involved in the suppression of ATP release from the urothelium, aiding visceral sensation of the urinary bladder. In the central pathways controlling the micturition reflex, 5-HT1A, 5-HT2A, and 5-HT7 are involved in regulation of bladder and urethral sphincter activities. Their functions, especially that of 5-HT1A, vary in a species- and site (spinal or supraspinal)- dependent manner. In addition to urinary bladder, 5-HT could be involved in prostate contraction and cell proliferation. Evidence indicates that 5-HT receptor subtypes may be novel therapeutic targets for lower urinary tract symptoms. FUNDING Grants-in-Aid for Scientific Research (C) (KAKENHI 23590707, 24590722, and 26460694) from the Japan Society for the Promotion of Science.
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Pau A, Catto M, Pinna G, Frau S, Murineddu G, Asproni B, Curzu MM, Pisani L, Leonetti F, Loza MI, Brea J, Pinna GA, Carotti A. Multitarget-Directed Tricyclic Pyridazinones as G Protein-Coupled Receptor Ligands and Cholinesterase Inhibitors. ChemMedChem 2015; 10:1054-70. [DOI: 10.1002/cmdc.201500124] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2015] [Indexed: 11/11/2022]
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14
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Novikov GV, Sivozhelezov VS, Shaitan KV. Influence of orthosteric ligand binding on the conformational dynamics of the β-2-adrenergic receptor via essential dynamics sampling simulation. Mol Biol 2014. [DOI: 10.1134/s0026893314030157] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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15
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GPCR & Company: Databases and Servers for GPCRs and Interacting Partners. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2014; 796:185-204. [DOI: 10.1007/978-94-007-7423-0_9] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
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16
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Labrecque P, Roy SJ, Fréchette L, Iorio-Morin C, Gallant MA, Parent JL. Inverse agonist and pharmacochaperone properties of MK-0524 on the prostanoid DP1 receptor. PLoS One 2013; 8:e65767. [PMID: 23762421 PMCID: PMC3677937 DOI: 10.1371/journal.pone.0065767] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2012] [Accepted: 05/01/2013] [Indexed: 01/09/2023] Open
Abstract
Prostaglandin D₂ (PGD₂) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gα(s)-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gα(i/o) proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD₂ induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.
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Affiliation(s)
- Pascale Labrecque
- Département de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada
- Centre de Recherche Clinique Étienne-Le Bel, Sherbrooke, Quebec, Canada
- Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada
| | - Sébastien J. Roy
- Département de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada
- Centre de Recherche Clinique Étienne-Le Bel, Sherbrooke, Quebec, Canada
- Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada
| | - Louis Fréchette
- Département de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada
- Centre de Recherche Clinique Étienne-Le Bel, Sherbrooke, Quebec, Canada
- Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada
| | - Christian Iorio-Morin
- Département de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada
- Centre de Recherche Clinique Étienne-Le Bel, Sherbrooke, Quebec, Canada
- Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada
| | - Maxime A. Gallant
- Département de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada
- Centre de Recherche Clinique Étienne-Le Bel, Sherbrooke, Quebec, Canada
- Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada
| | - Jean-Luc Parent
- Département de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada
- Centre de Recherche Clinique Étienne-Le Bel, Sherbrooke, Quebec, Canada
- Institut de Pharmacologie de Sherbrooke, Sherbrooke, Quebec, Canada
- * E-mail:
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17
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Novikov GV, Sivozhelezov VS, Shaitan KV. Study of structural dynamics of ligand-activated membrane receptors by means of principal component analysis. BIOCHEMISTRY (MOSCOW) 2013; 78:403-11. [PMID: 23590443 DOI: 10.1134/s0006297913040093] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The structural dynamics of three different ligand-activated G-protein coupled receptors (GPCRs) and the photoreactive receptor rhodopsin from mammals were comparatively studied. As a result, diagrams demonstrating the main structural differences between the studied membrane receptors were obtained. These diagrams represent the projection of the crystal structures of rhodopsin photointermediates and ligand-activated receptors onto the plane defined by the principal components. Thus, we were able to associate the activation process of the receptors with large-scale movements of their individual transmembrane (TM) domains. In addition, the dynamics of extracellular loops of ligand-activated receptors responsible for recognition and initial binding of ligands was studied. Based on these results, two parameters of functionally significant structural dynamics of membrane receptors can be thoroughly analyzed simultaneously - movements of individual TM helices and of extracellular loops.
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Affiliation(s)
- G V Novikov
- Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
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18
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Pradhan AA, Smith ML, Kieffer BL, Evans CJ. Ligand-directed signalling within the opioid receptor family. Br J Pharmacol 2013; 167:960-9. [PMID: 22708627 DOI: 10.1111/j.1476-5381.2012.02075.x] [Citation(s) in RCA: 115] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
The classic model of GPCR activation proposed that all agonists induce the same active receptor conformation. However, research over the last decade has shown that GPCRs exist in multiple conformations, and that agonists can stabilize different active states. The distinct receptor conformations induced by ligands result in distinct receptor-effector complexes, which produce varying levels of activation or inhibition of subsequent signalling cascades. This concept, referred to as ligand-directed signalling or biased agonism has important biological and therapeutic implications. Opioid receptors are G(i/o) GPCRs and regulate a number of important physiological functions, including pain, reward, mood, stress, gastrointestinal transport and respiration. A number of in vitro studies have shown biased agonism at the three opioid receptors (µ, δ and κ); however, in vivo consequences of this phenomenon have only recently been demonstrated. For the µ and δ opioid receptors, the majority of reported ligand selective behavioural effects are observed as differential adaptations to repeated drug administration. In terms of the κ opioid receptor, clear links between ligand-selective signalling events and specific in vivo responses have been recently characterized. Drugs for all three receptors are either already used or are being developed for clinical applications. There is clearly a need to better characterize the specific events that occur following agonist stimulation and how these relate to in vivo responses. This understanding could eventually lead to the development of tailor-made pharmacotherapies where advantageous drug effects can be selectively targeted over adverse effects.
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Affiliation(s)
- Amynah A Pradhan
- Semel Institute for Neuropsychiatry & Human Behavior, University of California Los Angeles, Los Angeles, CA 90024-1759, USA.
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19
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Allegretti M, Cesta MC, Garin A, Proudfoot AE. Current status of chemokine receptor inhibitors in development. Immunol Lett 2012; 145:68-78. [DOI: 10.1016/j.imlet.2012.04.003] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2011] [Accepted: 04/13/2012] [Indexed: 01/24/2023]
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20
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A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer. Nat Chem Biol 2012; 8:631-8. [PMID: 22634634 DOI: 10.1038/nchembio.962] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2011] [Accepted: 03/15/2012] [Indexed: 01/06/2023]
Abstract
Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gβγ but not Gα signaling triggered upon activation of Gα(i)-βγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gβγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.
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21
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Koener B, Focant MC, Bosier B, Maloteaux JM, Hermans E. Increasing the density of the D2L receptor and manipulating the receptor environment are required to evidence the partial agonist properties of aripiprazole. Prog Neuropsychopharmacol Biol Psychiatry 2012; 36:60-70. [PMID: 21871520 DOI: 10.1016/j.pnpbp.2011.08.007] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2011] [Revised: 08/05/2011] [Accepted: 08/10/2011] [Indexed: 11/30/2022]
Abstract
The clinical efficacy of aripiprazole in the treatment of psychosis relies on a partial agonism at D2 receptors. As the expression of this receptor differs physiologically between pre- and post-synaptic sites and is affected by pathological conditions or pharmacological treatments, it appears difficult to predict the clinical response to partial agonists. In addition, the response to this novel antipsychotic was shown to depend on the cell-line and the pathway analyzed, suggesting a functional selective profile at the D2 receptor. This study aims at examining the influence of receptor density and ionic environment on the pharmacological properties of aripiprazole. A cell line was developed in which the expression of the recombinant D2 receptor can be tightly manipulated using doxycycline and sodium butyrate. The potency and efficacy of aripiprazole and other reference D2 receptor ligands were examined in [35S]GTPγS binding assays, in buffers containing either NaCl or N-methyl-D-glucamine (NMDG) which is proposed to enhance G protein coupling. Increasing the density of D2 receptors considerably enhanced the [35S]GTPγS binding induced by dopamine and the full agonist NPA. In maximally induced cells, the agonist properties of the partial agonist (-)-3-PPP was revealed in a buffer containing NaCl, whereas the response to aripiprazole was not evidenced. Substituting NMDG for NaCl promoted the response to dopamine and (-)3-PPP and was proven efficient to reveal the partial agonist profile of aripiprazole. While NMDG substitution for NaCl strongly enhanced receptor-G protein coupling, these ionic manipulations are likely to influence receptor conformations, thereby modulating the activation of signaling pathways. Our data obtained with partial agonists acting at the D2 receptor suggest that these changes in the experimental conditions could contribute to reveal the functional selective profile of GPCR ligands. They also emphasize that the properties of functional selective ligands do not only depend on receptor density but also on the surrounding environment which likely differs between brain structures.
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Affiliation(s)
- Beryl Koener
- Institute of Neuroscience (Ions), Group of Neuropharmacology, Université Catholique de Louvain, Avenue Mounier 53, bte B1.53.02, B-1200 Brussels, Belgium
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22
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Fanelli F, De Benedetti PG. Update 1 of: computational modeling approaches to structure-function analysis of G protein-coupled receptors. Chem Rev 2011; 111:PR438-535. [PMID: 22165845 DOI: 10.1021/cr100437t] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Francesca Fanelli
- Dulbecco Telethon Institute, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy.
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23
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Delta opioid receptor analgesia: recent contributions from pharmacology and molecular approaches. Behav Pharmacol 2011; 22:405-14. [PMID: 21836459 DOI: 10.1097/fbp.0b013e32834a1f2c] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Delta opioid receptors represent a promising target for the development of novel analgesics. A number of tools have been developed recently that have significantly improved our knowledge of δ receptor function in pain control. These include several novel δ agonists with potent analgesic properties, and genetic mouse models with targeted mutations in the δ opioid receptor gene. Also, recent findings have further documented the regulation of δ receptor function at cellular level, which impacts on the pain-reducing activity of the receptor. These regulatory mechanisms occur at transcriptional and post-translational levels, along agonist-induced receptor activation, signaling and trafficking, or in interaction with other receptors and neuromodulatory systems. All these tools for in-vivo research, and proposed mechanisms at molecular level, have tremendously increased our understanding of δ receptor physiology, and contribute to designing innovative strategies for the treatment of chronic pain and other diseases such as mood disorders.
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24
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Salon JA, Lodowski DT, Palczewski K. The significance of G protein-coupled receptor crystallography for drug discovery. Pharmacol Rev 2011; 63:901-37. [PMID: 21969326 PMCID: PMC3186081 DOI: 10.1124/pr.110.003350] [Citation(s) in RCA: 160] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.
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Affiliation(s)
- John A Salon
- Department of Molecular Structure, Amgen Incorporated, Thousand Oaks, California, USA
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25
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Blaho VA, Hla T. Regulation of mammalian physiology, development, and disease by the sphingosine 1-phosphate and lysophosphatidic acid receptors. Chem Rev 2011; 111:6299-320. [PMID: 21939239 PMCID: PMC3216694 DOI: 10.1021/cr200273u] [Citation(s) in RCA: 121] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Affiliation(s)
- Victoria A. Blaho
- Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY 10065
| | - Timothy Hla
- Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY 10065
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26
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Pradhan AA, Befort K, Nozaki C, Gavériaux-Ruff C, Kieffer BL. The delta opioid receptor: an evolving target for the treatment of brain disorders. Trends Pharmacol Sci 2011; 32:581-90. [PMID: 21925742 DOI: 10.1016/j.tips.2011.06.008] [Citation(s) in RCA: 218] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2011] [Revised: 06/18/2011] [Accepted: 06/21/2011] [Indexed: 12/14/2022]
Abstract
Compared to the better-known mu opioid receptor, delta opioid receptors have been relatively understudied. However, the development of highly selective delta opioid agonists and the availability of genetic mouse models have extended our knowledge of delta opioid receptors in vivo. Here we review recent developments in the characterization of delta opioid receptor biology and aspects of delta opioid receptor function that have potential for therapeutic targeting. Preclinical data have confirmed that delta opioid receptor activation reduces persistent pain and improves negative emotional states; clinical trials have been initiated to assess the effectiveness of delta opioid agonists in chronic pain and depression. Furthermore, a possible role for these receptors in neuroprotection is being investigated. The usefulness of targeting delta opioid receptors in drug abuse remains open and a role for these receptors in impulse control disorders is emerging. Finally, the recent demonstration of biased agonism at the delta opioid receptor in vivo opens novel perspectives towards targeting specific therapeutic effects through drug design.
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Affiliation(s)
- Amynah A Pradhan
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch, France
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27
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CODA-RET reveals functional selectivity as a result of GPCR heteromerization. Nat Chem Biol 2011; 7:624-30. [PMID: 21785426 PMCID: PMC3158273 DOI: 10.1038/nchembio.623] [Citation(s) in RCA: 99] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2010] [Accepted: 05/16/2011] [Indexed: 01/09/2023]
Abstract
Here we present a novel method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 receptor (D2R) agonist R(–)-Propylnorapomorphine (NPA) to change the Gαi conformation via the D2R protomer in the D1-D2 heteromer was enhanced 10-fold relative to that observed in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity, in which a drug acts differently at a GPCR protomer depending on the identity of the second protomer that participates in forming the signaling unit, opening the door to enhanced pharmacological specificity through targeting differences between homomeric and heteromeric signaling.
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Abstract
Increasing numbers of compounds, previously classified as antagonists, were shown to inhibit this spontaneous or constitutive receptor activity, instead of leave it unaffected as expected for a formal antagonist. In addition, some other antagonists did not have any effect by themselves, but prevented the inhibition of constitutive activity induced by thought-to-be antagonists. These thought-to-be antagonists with negative efficacy are now known as "inverse agonists." Inverse agonism at βAR has been evidenced for both subtypes in wild-type GPCRs systems and in engineered systems with high constitutive activity. It is important to mention that native systems are of particular importance for analyzing the in vivo relevance of constitutive activity because these systems have physiological expression levels of target receptors. Studies of inverse agonism of β blockers in physiological setting have also evidenced that pathophysiological conditions can affect pharmacodynamic properties of these ligands. To date, hundreds of clinically well-known drugs have been tested and classified for this property. Prominent examples include the beta-blockers propranolol, alprenolol, pindolol, and timolol used for treating hypertension, angina pectoris, and arrhythmia that act on the β₂ARs, metoprolol, and bisoprolol used for treating hypertension, coronary heart disease, and arrhythmias by acting on β₁ARs. Inverse agonists seem to be useful in the treatment of chronic disease characterized by harmful effects resulting from β₁AR and β₂AR overactivation, such as heart failure and asthma, respectively.
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Affiliation(s)
- Carlos A Taira
- Cátedra de Farmacología, Instituto de Fisiopatología y Bioquímica Clínica, Universidad de Buenos Aires, CONICET, Junín 956, Buenos Aires, Argentina
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29
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Abstract
With the emergence of information describing functional selectivity and biased agonists and antagonists has come a lack of confidence in "one size fits all" assays for detection of agonism. Seven-transmembrane receptors are pleiotropic with respect to the signaling protein to which they couple in a cell, and many conformations of the receptor can be formed; this leads to systems where ligands can stabilize unique conformations that go on to selectively activate signaling pathways. Thus, such "biased" ligands can produce cell-specific agonism that may require targeted assays to detect and quantify. It also predicts that ligands can have many different efficacies for the many behaviors that the receptor can exhibit (referred to as "pluridimensional efficacy"), leading to a breakdown in the common classifications of agonist and antagonist. This all poses unique challenges to the pharmacologic nomenclature of drugs, the detection and optimization of new drugs, and the association of phenotypic clinical profiles with pharmacological properties of drugs.
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Affiliation(s)
- Terry Kenakin
- Platform Technology Sciences, GlaxoSmithKline Research and Development, Research Triangle Park, NC 27709, USA.
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30
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Ligand-directed trafficking of the δ-opioid receptor in vivo: two paths toward analgesic tolerance. J Neurosci 2011; 30:16459-68. [PMID: 21147985 DOI: 10.1523/jneurosci.3748-10.2010] [Citation(s) in RCA: 113] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80-but not ARM390-administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca²+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of biased agonism in vivo.
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31
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Schröder R, Janssen N, Schmidt J, Kebig A, Merten N, Hennen S, Müller A, Blättermann S, Mohr-Andrä M, Zahn S, Wenzel J, Smith NJ, Gomeza J, Drewke C, Milligan G, Mohr K, Kostenis E. Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat Biotechnol 2010; 28:943-9. [PMID: 20711173 DOI: 10.1038/nbt.1671] [Citation(s) in RCA: 219] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms.
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Affiliation(s)
- Ralf Schröder
- Molecular, Cellular, and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Bonn, Germany
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32
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Kenakin T, Miller LJ. Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery. Pharmacol Rev 2010; 62:265-304. [PMID: 20392808 PMCID: PMC2879912 DOI: 10.1124/pr.108.000992] [Citation(s) in RCA: 464] [Impact Index Per Article: 30.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
It is useful to consider seven transmembrane receptors (7TMRs) as disordered proteins able to allosterically respond to a number of binding partners. Considering 7TMRs as allosteric systems, affinity and efficacy can be thought of in terms of energy flow between a modulator, conduit (the receptor protein), and a number of guests. These guests can be other molecules, receptors, membrane-bound proteins, or signaling proteins in the cytosol. These vectorial flows of energy can yield standard canonical guest allostery (allosteric modification of drug effect), effects along the plane of the cell membrane (receptor oligomerization), or effects directed into the cytosol (differential signaling as functional selectivity). This review discusses these apparently diverse pharmacological effects in terms of molecular dynamics and protein ensemble theory, which tends to unify 7TMR behavior toward cells. Special consideration will be given to functional selectivity (biased agonism and biased antagonism) in terms of mechanism of action and potential therapeutic application. The explosion of technology that has enabled observation of diverse 7TMR behavior has also shown how drugs can have multiple (pluridimensional) efficacies and how this can cause paradoxical drug classification and nomenclatures.
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Affiliation(s)
- Terry Kenakin
- GlaxoSmithKline, 5 Moore Drive, Mailtstop V-287, Research Triangle Park, NC 27709, USA.
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33
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Bosier B, Muccioli GG, Hermans E, Lambert DM. Functionally selective cannabinoid receptor signalling: therapeutic implications and opportunities. Biochem Pharmacol 2010; 80:1-12. [PMID: 20206137 DOI: 10.1016/j.bcp.2010.02.013] [Citation(s) in RCA: 136] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2009] [Revised: 02/17/2010] [Accepted: 02/24/2010] [Indexed: 10/19/2022]
Abstract
The CB(1) and CB(2) cannabinoid receptors are G protein-coupled receptors (GPCRs) recognized by a variety of endogenous ligands and activating multiple signalling pathways. This multiplicity of ligands and intracellular transduction mechanisms supports a complex control of physiological functions by the endocannabinoid system, but requires a finely tuned regulation of the signalling events triggered on receptor activation. Here we review the diverse signalling pathways activated by the cannabinoid receptors and discuss the mechanisms allowing for specificity in the associated functional responses triggered by endogenous or exogenous ligands. At variance with the classical concept that all agonists at a given GPCR induce a similar repertoire of downstream events in all tissues, we also summarize the experimental evidence supporting the existence of functional selectivity and protean agonism at cannabinoid receptors. By placing emphasis on the ligand- or constitutive activity-dependent specifications of receptor-G protein coupling, these concepts explain how distinct cannabinoid ligands may activate specific downstream mediators. Finally, although both the diversity and specificity in cannabinoid signalling are now established in vitro, few data are available from in vivo studies. Therefore, we conclude this review by examining the experimental evidence supporting the physiological relevance of this complexity in the cannabinoid system. The ability to selectively manipulate physiological functions, through activation of defined signalling cascades, will in all likelihood help in the development of efficacious and safe cannabinoid-based therapeutics for a variety of indications.
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Affiliation(s)
- Barbara Bosier
- Unité de Chimie Pharmaceutique et de Radiopharmacie (CMFA 7340), Louvain Drug Research Institute, Brussels, Belgium
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Dufton N, Hannon R, Brancaleone V, Dalli J, Patel HB, Gray M, D'Acquisto F, Buckingham JC, Perretti M, Flower RJ. Anti-inflammatory role of the murine formyl-peptide receptor 2: ligand-specific effects on leukocyte responses and experimental inflammation. THE JOURNAL OF IMMUNOLOGY 2010; 184:2611-2619. [PMID: 20107188 DOI: 10.4049/jimmunol.0903526] [Citation(s) in RCA: 251] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The human formyl-peptide receptor (FPR)-2 is a G protein-coupled receptor that transduces signals from lipoxin A(4), annexin A1, and serum amyloid A (SAA) to regulate inflammation. In this study, we report the creation of a novel mouse colony in which the murine FprL1 FPR2 homologue, Fpr2, has been deleted and describe its use to explore the biology of this receptor. Deletion of murine fpr2 was verified by Southern blot analysis and PCR, and the functional absence of the G protein-coupled receptor was confirmed by radioligand binding assays. In vitro, Fpr2(-/-) macrophages had a diminished response to formyl-Met-Leu-Phe itself and did not respond to SAA-induced chemotaxis. ERK phosphorylation triggered by SAA was unchanged, but that induced by the annexin A1-derived peptide Ac2-26 or other Fpr2 ligands, such as W-peptide and compound 43, was attenuated markedly. In vivo, the antimigratory properties of compound 43, lipoxin A(4), annexin A1, and dexamethasone were reduced notably in Fpr2(-/-) mice compared with those in wild-type littermates. In contrast, SAA stimulated neutrophil recruitment, but the promigratory effect was lost following Fpr2 deletion. Inflammation was more marked in Fpr2(-/-) mice, with a pronounced increase in cell adherence and emigration in the mesenteric microcirculation after an ischemia-reperfusion insult and an augmented acute response to carrageenan-induced paw edema, compared with that in wild-type controls. Finally, Fpr2(-/-) mice exhibited higher sensitivity to arthrogenic serum and were completely unable to resolve this chronic pathology. We conclude that Fpr2 is an anti-inflammatory receptor that serves varied regulatory functions during the host defense response. These data support the development of Fpr2 agonists as novel anti-inflammatory therapeutics.
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Affiliation(s)
- Neil Dufton
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
| | - Robert Hannon
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
| | - Vincenzo Brancaleone
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
| | - Jesmond Dalli
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
| | - Hetal B Patel
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
| | - Mohini Gray
- Medical Research Council Centre for Inflammation, Edinburgh, United Kingdom
| | - Fulvio D'Acquisto
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
| | | | - Mauro Perretti
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
| | - Roderick J Flower
- The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London
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Cherezov V, Abola E, Stevens RC. Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets. Methods Mol Biol 2010; 654:141-68. [PMID: 20665265 PMCID: PMC2973844 DOI: 10.1007/978-1-60761-762-4_8] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/16/2023]
Abstract
G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using an LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment, extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.
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Affiliation(s)
- Vadim Cherezov
- Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA
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Hudson BD, Hébert TE, Kelly MEM. Ligand- and heterodimer-directed signaling of the CB(1) cannabinoid receptor. Mol Pharmacol 2010; 77:1-9. [PMID: 19837905 DOI: 10.1124/mol.109.060251] [Citation(s) in RCA: 85] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2025] Open
Abstract
Seven-transmembrane G protein-coupled receptors (GPCRs) represent the single largest family of cell surface receptors. Signaling through these receptors is controlled by changes in the conformation of the receptor from inactive to active conformations, which in turn lead to the activation of multiple downstream signaling pathways. To facilitate greater diversity in signaling responses, many of these receptors are capable of adopting several distinct active conformations, in which each couples preferentially to its own set of downstream signaling partners. Because these unique signaling responses result from specific receptor active conformations, GPCR signaling may be directed toward these selective responses through either strength-of-signal effects resulting from partial agonism or through biased agonism and functional selectivity, resulting from the selective stabilization of one active conformation over the others. This review uses the CB(1) cannabinoid receptor as a specific example to highlight the contribution of two important aspects of GPCR function-orthosteric ligand binding and receptor heterodimerization-toward directed GPCR signaling.
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Affiliation(s)
- Brian D Hudson
- Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 1X5
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Leduc M, Breton B, Galés C, Le Gouill C, Bouvier M, Chemtob S, Heveker N. Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands. J Pharmacol Exp Ther 2009; 331:297-307. [PMID: 19584306 DOI: 10.1124/jpet.109.156398] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Classically, the prostaglandin E(2) (PGE(2)) receptor EP(4) has been classified as coupling to the Galpha(s) subunit, leading to intracellular cAMP increases. However, EP(4) signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Galpha(i) proteins and beta-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP(4) ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP(4) ligands for activation of Galpha(s), Galpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE(2) was the most selective in activating Galpha(s), whereas PGF(2alpha) and PGE(1) alcohol were the most biased for activating Galpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and Galpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2alpha), PGD(2), or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE(2) analogs in experimental and possibly clinical settings, because their activity spectra on EP(4) differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP(4) agonists should be applicable for the study of other GPCRs.
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Affiliation(s)
- Martin Leduc
- Department of Biochemistry, Université de Montréal, Québec, Canada
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Whyte LS, Ryberg E, Sims NA, Ridge SA, Mackie K, Greasley PJ, Ross RA, Rogers MJ. The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo. Proc Natl Acad Sci U S A 2009; 106:16511-6. [PMID: 19805329 PMCID: PMC2737440 DOI: 10.1073/pnas.0902743106] [Citation(s) in RCA: 235] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2009] [Indexed: 11/18/2022] Open
Abstract
GPR55 is a G protein-coupled receptor recently shown to be activated by certain cannabinoids and by lysophosphatidylinositol (LPI). However, the physiological role of GPR55 remains unknown. Given the recent finding that the cannabinoid receptors CB(1) and CB(2) affect bone metabolism, we examined the role of GPR55 in bone biology. GPR55 was expressed in human and mouse osteoclasts and osteoblasts; expression was higher in human osteoclasts than in macrophage progenitors. Although the GPR55 agonists O-1602 and LPI inhibited mouse osteoclast formation in vitro, these ligands stimulated mouse and human osteoclast polarization and resorption in vitro and caused activation of Rho and ERK1/2. These stimulatory effects on osteoclast function were attenuated in osteoclasts generated from GPR55(-/-) macrophages and by the GPR55 antagonist cannabidiol (CBD). Furthermore, treatment of mice with this non-psychoactive constituent of cannabis significantly reduced bone resorption in vivo. Consistent with the ability of GPR55 to suppress osteoclast formation but stimulate osteoclast function, histomorphometric and microcomputed tomographic analysis of the long bones from male GPR55(-/-) mice revealed increased numbers of morphologically inactive osteoclasts but a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. These data reveal a role of GPR55 in bone physiology by regulating osteoclast number and function. In addition, this study also brings to light an effect of both the endogenous ligand, LPI, on osteoclasts and of the cannabis constituent, CBD, on osteoclasts and bone turnover in vivo.
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MESH Headings
- Animals
- Animals, Newborn
- Bone Density
- Bone Resorption/prevention & control
- Bone and Bones/cytology
- Bone and Bones/metabolism
- Cannabidiol/pharmacology
- Cell Line, Tumor
- Cells, Cultured
- Dose-Response Relationship, Drug
- Female
- Fluorescent Antibody Technique
- Humans
- Lysophospholipids/pharmacology
- Male
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Osteoblasts/cytology
- Osteoblasts/drug effects
- Osteoblasts/metabolism
- Osteoclasts/cytology
- Osteoclasts/drug effects
- Osteoclasts/metabolism
- Osteogenesis/drug effects
- Receptors, Cannabinoid/genetics
- Receptors, Cannabinoid/metabolism
- Receptors, G-Protein-Coupled/genetics
- Receptors, G-Protein-Coupled/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
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Affiliation(s)
- Lauren S. Whyte
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom
| | | | | | - Susan A. Ridge
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom
| | - Ken Mackie
- Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47401
| | | | - Ruth A. Ross
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom
| | - Michael J. Rogers
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom
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Brea J, Castro M, Giraldo J, López-Giménez JF, Padín JF, Quintián F, Cadavid MI, Vilaró MT, Mengod G, Berg KA, Clarke WP, Vilardaga JP, Milligan G, Loza MI. Evidence for distinct antagonist-revealed functional states of 5-hydroxytryptamine(2A) receptor homodimers. Mol Pharmacol 2009; 75:1380-91. [PMID: 19279328 DOI: 10.1124/mol.108.054395] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The serotonin (5-hydroxytryptamine; 5-HT) 2A receptor is a cell surface class A G protein-coupled receptor that regulates a multitude of physiological functions of the body and is a target for antipsychotic drugs. Here we found by means of fluorescence resonance energy transfer and immunoprecipitation studies that the 5-HT(2A)-receptor homodimerized in live cells, which we linked with its antagonist-dependent fingerprint in both binding and receptor signaling. Some antagonists, like the atypical antipsychotics clozapine and risperidone, differentiate themselves from others, like the typical antipsychotic haloperidol, antagonizing these 5-HT(2A) receptor-mediated functions in a pathway-specific manner, explained here by a new model of multiple active interconvertible conformations at dimeric receptors.
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Affiliation(s)
- José Brea
- Departamento de Farmacología, Instituto de Farmacia Industrial, Universidad de Santiago de Compostela, Spain
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Bosier B, Lambert DM, Hermans E. Reciprocal influences of CB1 cannabinoid receptor agonists on ERK and JNK signalling in N1E-115 cells. FEBS Lett 2008; 582:3861-7. [PMID: 18950629 DOI: 10.1016/j.febslet.2008.10.022] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2008] [Revised: 10/03/2008] [Accepted: 10/15/2008] [Indexed: 11/30/2022]
Abstract
Agonists acting at the CB1 cannabinoid receptor in N1E-115 neuroblastoma cells were found to activate MAPK family members with reciprocal efficacies. Thus, HU 210 robustly increased phosphorylation of ERK1/2 whereas CP 55,940 was more effective in activating JNK. The use of selected kinase inhibitors confirmed that distinct signalling cascades were involved in these responses. This reciprocal control of MAPK activity was correlated with the observation that HU 210- and CP 55,940-mediated regulations of tyrosine hydroxylase gene expression were respectively impaired by MEK and JNK inhibitors. These data indicate that complex interactions of the CB1 receptor with intracellular signalling partners controlling MAPK activities may explain the apparent disparities in cellular responses to functional selective agonists.
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Affiliation(s)
- Barbara Bosier
- Unité de Chimie Pharmaceutique et de Radiopharmacie (UCL 7340), Université catholique de Louvain, 73 40, Av E.Mounier, B-1200 Brussels, Belgium
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Bosier B, Hermans E, Lambert DM. Concomitant activation of adenylyl cyclase suppresses the opposite influences of CB(1) cannabinoid receptor agonists on tyrosine hydroxylase expression. Biochem Pharmacol 2008; 77:216-27. [PMID: 18992715 DOI: 10.1016/j.bcp.2008.10.010] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2008] [Revised: 10/03/2008] [Accepted: 10/08/2008] [Indexed: 11/26/2022]
Abstract
The CB(1) cannabinoid receptor shows complex interactions with intracellular signalling partners, and responses to cannabinoid ligands are likely to be influenced by concomitant inputs modifying the overall tone of signalling cascades. This appears even more relevant as we previously evidenced opposite regulations of tyrosine hydroxylase (TH) expression by the two common cannabinoid agonists HU 210 and CP 55,940. Therefore, we studied the consequences of manipulating adenylyl cyclase activity with forskolin on the regulation of TH gene transcription in neuroblastoma cells (N1E-115). Reporter gene experiments performed with the luciferase sequence cloned under the control of modified fragments of the TH gene promoter revealed that the AP-1 consensus sequence is essential for cannabinoid-mediated regulation of TH expression. Consistently, inhibition of PKC totally blocked the responses mediated by both HU 210 and CP 55,940. In addition, forskolin which boosts adenylyl cyclase activity remarkably modified the responses to the cannabinoid agonists. Thus, in these conditions, both agonists efficiently reduced TH gene promoter activity, a response requiring functional PKA/CRE-dependent signallings. Finally, the modulations of the promoter were inhibited in pertussis toxin treated cells, suggesting that responses to both agonists are mediated through G(i/o)-dependent mechanisms. Emphasising on the importance of functional selectivity at GPCRs, these data demonstrate that the concomitant activation of adenylyl cyclase by forskolin strongly influences the biochemical responses triggered by distinct cannabinoid agonists. Together our results suggest that the physiological modulation of TH expression by cannabinoid agonists in dopaminergic neurons would be influenced by additional endogenous inputs.
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Affiliation(s)
- Barbara Bosier
- Unité de Chimie Pharmaceutique et de Radiopharmacie, Université catholique de Louvain, Brussels, Belgium
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Membrane signalling complexes: implications for development of functionally selective ligands modulating heptahelical receptor signalling. Cell Signal 2008; 21:179-85. [PMID: 18790047 DOI: 10.1016/j.cellsig.2008.08.013] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2008] [Accepted: 08/24/2008] [Indexed: 11/24/2022]
Abstract
Technological development has considerably changed the way in which we evaluate drug efficacy and has led to a conceptual revolution in pharmacological theory. In particular, molecular resolution assays have revealed that heptahelical receptors may adopt multiple active conformations with unique signalling properties. It is therefore becoming widely accepted that ligand ability to stabilize receptor conformations with distinct signalling profiles may allow to direct the stimulus generated by an activated receptor towards a specific signalling pathway. This capacity to induce only a subset of the ensemble of responses regulated by a given receptor has been termed "functional selectivity" (or "stimulus trafficking"), and provides the bases for a highly specific regulation of receptor signalling. Concomitant with these observations, heptahelical receptors have been shown to associate with G proteins and effectors to form multimeric arrays. These complexes are constitutively formed during protein synthesis and are targeted to the cell surface as integral signalling units. Herein we summarize evidence supporting the existence of such constitutive signalling arrays and analyze the possibility that they may constitute viable targets for developing ligands with "functional selectivity".
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Differential modulation of AP-1- and CRE-driven transcription by cannabinoid agonists emphasizes functional selectivity at the CB1 receptor. Br J Pharmacol 2008; 155:24-33. [PMID: 18536748 DOI: 10.1038/bjp.2008.230] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND AND PURPOSE Long-term adaptations to pharmacological stimuli frequently originate from modulation of complex intracellular signalling pathways. We previously reported that HU210 and CP55940, two CB1 cannabinoid receptor agonists, induced opposite effects on TH expression. Herein, we characterized their influence on cAMP response element (CRE) and activator protein 1 (AP-1)-mediated regulation of gene transcription. EXPERIMENTAL APPROACH The activity of the agonists was examined on transfected N1E-115 cells in which expression of the luciferase reporter gene was controlled by transcription promoters consisting of repeats of either CRE or AP-1 elements. In addition, the implication of classical signalling pathways was investigated using a variety of kinase inhibitors. KEY RESULTS Consistent with the CB1-mediated reduction of cAMP accumulation, both ligands decreased CRE-driven luciferase expression with similar potencies. HU210 also exhibited a concentration-dependent reduction of luciferase activity in cells engineered to examine AP-1-controlled transcription, whereas such response was not obtained with CP55940. Responses were all inhibited by SR141716A and were modified in Pertussis toxin-treated cells, suggesting agonist-selective regulations of distinct Gi/o-dependent mechanisms through CB1 receptor activation. Finally, PKC inhibitors efficiently inhibited the paradoxical effect of HU210 on AP-1-mediated transcription, indicating selective regulation of PKC-dependent responses. CONCLUSIONS AND IMPLICATIONS Together, our results demonstrate that two cannabinoid ligands, commonly used as reference agonists acting on the same receptor with similar affinities, differentially modulate gene transcription through distinct controls of AP-1. This could reflect activation of distinct subsets of Gi/o-proteins, supporting the concept of functional selectivity at CB1 receptors.
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Affiliation(s)
- David E Nichols
- Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue University, West Lafayette, Indiana 47906-2091, USA.
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Canals M, Milligan G. Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors. J Biol Chem 2008; 283:11424-34. [PMID: 18319252 DOI: 10.1074/jbc.m710300200] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.
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Affiliation(s)
- Meritxell Canals
- Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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47
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Penn RB. Embracing emerging paradigms of G protein-coupled receptor agonism and signaling to address airway smooth muscle pathobiology in asthma. Naunyn Schmiedebergs Arch Pharmacol 2008; 378:149-69. [PMID: 18278482 DOI: 10.1007/s00210-008-0263-1] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2007] [Accepted: 01/15/2008] [Indexed: 01/04/2023]
Abstract
G protein-coupled receptors (GPCRs) regulate numerous airway cell functions, and signaling events transduced by GPCRs are important in both asthma pathogenesis and therapy. Indeed, most asthma therapies target GPCRs either directly or indirectly. Within recent years, our understating of how GPCRs signal and are regulated has changed significantly as new concepts have emerged and traditional ideas have evolved. In this review, we discuss current concepts regarding constitutive GPCR activity and receptor agonism, functional selectivity, compartmentalized signaling, and GPCR desensitization. We further discuss the relevance of these ideas to asthma and asthma therapy, while emphasizing their potential application to the GPCR signaling in airway smooth muscle that regulates airway patency and thus disease severity.
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Affiliation(s)
- Raymond B Penn
- Department of Internal Medicine, Wake Forest University Health Sciences Center, Winston-Salem, NC 27157, USA.
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Abstract
This commentary discusses a paper in this issue by Dr Jillian Baker on the antagonism of histamine H(2) receptors. It is an excellent example of the use of pharmacological principles to determine what systems can and can't do from the point of view of agonist-dependent antagonism. The most common model of antagonism, namely orthosteric, cannot discern agonist type; i.e. all agonists are blocked equally by a given orthosteric antagonist. Therefore, if quantitative assessment of antagonism unveils agonist dependence, then this is something an orthosteric mechanism cannot do and another mechanism must be considered. A simple alternative is a permissive allosteric model whereby the agonist and antagonist interact through conformational changes in the receptor protein. Under these circumstances, an agonist-antagonist dialogue can ensue whereby the nature of the agonist determines the magnitude of antagonist effect. Jillian Baker contrasts antagonist systems with historical data obtained for beta-adrenoceptors and the present data for histamine H(2) receptors where the simpler model of orthosteric antagonism suffices and thus shows how quantitative receptor pharmacology can be used to determine the molecular mechanism of antagonism.
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Affiliation(s)
- T Kenakin
- GlaxoSmithKline Research and Development, Research Triangle Park, NC 27709, USA.
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49
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Abstract
In contrast to earlier concepts, it seems that distinct ligands acting on the same receptor may elicit qualitative different response patterns, a phenomenon given many names, including "functional selectivity," "agonist-directed trafficking," "biased agonism," "protean agonism," or "ligand-directed signaling." In this issue of Molecular Pharmacology, Sato et al. (p. 1359) extend this concept to beta(3)-adrenergic receptors and report that distinct ligands can activate a single distal response via different signaling pathways. Moreover, they demonstrate that expression density can affect how distinct ligands acting on the same receptor differentially induce cellular responses. We discuss the underlying concepts for such findings and their implications for drug discovery.
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Affiliation(s)
- Martin C Michel
- Dept. Pharmacology and Pharmacotherapy, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, Netherlands.
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