DOCSLIB.ORG

Development and Validation of a Novel Quantitative Assay for Cell

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Development and Validation of a Novel Quantitative Assay for Cell Development and Validation of a Novel Quantitative Assay for Cell Surface expression of GPCRs using a Receptor β-lactamase Fusion Protein and the Colourometric Substrate Nitrocefin by Vincent Lam A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Pharmacology and Toxicology University of Toronto © Copyright by Vincent Lam 2013 Development and Validation of a Novel Quantitative Assay for Cell Surface Expression of GPCRs using a Receptor β-lactamase Fusion Protein and the Colourometric Substrate Nitrocefin Vincent Lam Master of Science Department of Pharmacology and Toxicology University of Toronto 2013 Abstract Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA. ii Acknowledgments I would like to thank the following people who have made completion of the thesis possible. First I would like to express my thanks and gratitude to Dr. Ali Salahpour for his guidance, mentorship, support, and endless positive encouragement throughout the completion of this thesis. I would also like to thank Dr. Jane Mitchell (co-supervisor) for her advice and critical analyses of my work. I would also like to thank Dr. Amy Ramsey and Dr. David Riddick (Advisor) for their support, guidance, and suggestions. Furthermore I would like to thank the members of the Ramsey and Salahpour lab for their support and intellectual discussion during my studies. Lastly I would like to thank my friends and family for their endless encouragement and support throughout my time in graduate studies, without them this thesis would not have been possible. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Abbreviations ................................................................................................................... viii List of Appendices .......................................................................................................................... x Chapter 1 Introduction .................................................................................................................... 1 Specific Aims and Working Hypothesis ......................................................................................... 1 1.0 GPCR Pharmacology .......................................................................................................... 2 1.1 GPCR Classification ........................................................................................................... 3 1.2 Structure and function ......................................................................................................... 4 1.3 GPCR Signalling ................................................................................................................. 5 1.3.1 Ligand binding to receptor ...................................................................................... 5 1.3.2 G-proteins ............................................................................................................... 7 1.3.3 Effectors .................................................................................................................. 8 1.4 GPCR Trafficking from the ER .......................................................................................... 9 1.4.1 Signal Sequences .................................................................................................. 10 1.4.2 Post translational modifications ........................................................................... 12 1.4.3 Molecular Chaperones .......................................................................................... 14 1.4.4 Pharmacological Chaperones and Diseases ......................................................... 16 1.5 GPCR Oligomerization ..................................................................................................... 17 1.6 GPCR Endocytic Trafficking ............................................................................................ 18 1.6.1 G-protein Coupled Receptor Kinases (GRK) ....................................................... 19 1.6.2 Arrestins ................................................................................................................ 20 1.6.3 Tonic/Constitutive Internalization ........................................................................ 22 1.6.4 Endocytic pathway ................................................................................................ 22 1.7 Summary of Assays for measuring surface expression of GPCRs ................................... 23 1.7.1 Fluorogen Activating Protein Biosensor ............................................................... 25 1.7.2 Internalization Assays ........................................................................................... 26 1.7.3 N-terminal GPCR Fusion Tags ............................................................................. 27 1.8 β-lactamase Assay ............................................................................................................. 28 Chapter 2 Materials and Methods ................................................................................................. 33 2.1 Reagents ............................................................................................................................ 33 2.2 Plasmid Construction ........................................................................................................ 33 2.3 Cell Culture ....................................................................................................................... 33 2.4 Generation of Stable Cell Lines and Transient Transfections .......................................... 34 2.5 Western Blotting ............................................................................................................... 34 2.6 βlac-β2AR Immunofluorescence ...................................................................................... 34 2.7 βlac-β2AR Functional Assay using BRET EPAC cAMP Biosensor ............................... 35 2.8 βlac Assay ......................................................................................................................... 35 2.9 ELISA ............................................................................................................................... 35 2.10 βlac-β2AR Agonist Studies............................................................................................... 36 2.11 βlac-β2AR Antagonist Studies .......................................................................................... 36 iv 2.12 βlac-β2AR Z’ Determination for Agonist Induced Internalization................................... 36 2.13 GBR1 Molecular Chaperoning Studies ............................................................................ 36 2.14 Data Analyses ................................................................................................................... 37 Chapter 3 Results .......................................................................................................................... 38 3.1 Generation of the βlac Plasmids and Stable Cell Lines Expressing βlac-GPCR Fusion Constructs ................................................................................................................................. 38 3.2 Trafficking and Signalling of the βlac-β2AR ................................................................... 38 3.3 βlac-β2AR experiments .................................................................................................... 42 3.3.1 Comparison of Isoproterenol stimulated β2AR Internalization using the βlac and the ELISA Assays ...................................................................................................... 42 3.3.2 Antagonist Blocking of Isoproterenol Induced Internalization of the β2AR ........ 46 3.3.3 Z’ Determination of the SS-HA-βlac-β2AR Internalization ................................. 46 3.3.4 Pharmacological Chaperoning using β2AR Antagonists ...................................... 48 3.4 Comparison of
Load more

Recommended publications
  • REVIEW Dimerization and Oligomerization of G-Protein
    435 REVIEW Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions La´szlo´ Szidonya1, Miklo´s Cserzo˝ 1 and La´szlo´ Hunyady1,2 1Department of Physiology, Semmelweis University, PO Box 259, H-1444 Budapest, Hungary 2Laboratory for Neurobiochemistry and Molecular Physiology, Hungarian Academy of Sciences and Semmelweis University, H-1444 Budapest, Hungary (Correspondence should be addressed to L Hunyady; Email: [email protected]) Abstract Dimerization or oligomerization of G-protein-coupled homo- or heterodimeric or oligomeric complexes, in which receptors (GPCRs) is a novel concept, which may lead to receptor monomers have stable direct interactions. However, the reevaluation of the actions of pharmacological ligands, overwhelming amounts of data suggest that many GPCRs hormones, neurotransmitters, and other mediators acting on exhibit functional properties that require direct or indirect GPCRs. Although a large number of data obtained using interactions between clustered receptors. Although it is different biophysical, biochemical and structural methods, difficult to conclude, about the exact nature of these and functional approaches argue for dimerization or interactions, dimerization or oligomerization of GPCRs is a oligomerization of these receptors, several publications useful paradigm for pharmacologists to study properties of criticized the applied methods and challenged the concept. receptors, which require functionally important clustering of The aim of this paper is to review the data that support the receptors, such as trafficking of newly synthesized receptors to concept of receptor oligomerization, and the most important the cell surface, allosteric modulation of ligand binding, arguments against it. We conclude that it will require major signaling specificity, co-internalization, or cross-inhibition of methodical improvements to obtain decisive proof, whether GPCRs.
    [Show full text]
  • Cell Surface Targeting of ␮-␦ Opioid Receptor Heterodimers by RTP4
    Cell surface targeting of ␮-␦ opioid receptor heterodimers by RTP4 Fabien M. De´ caillot, Raphael Rozenfeld, Achla Gupta, and Lakshmi A. Devi* Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029 Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved August 22, 2008 (received for review April 29, 2008) ␮ opioid receptors are G protein–coupled receptors that mediate heterodimers, respectively. These findings, together with the the pain-relieving effects of clinically used analgesics, such as observation that the development of tolerance correlates with morphine. Accumulating evidence shows that ␮-␦ opioid het- the enhanced surface expression (through externalization) of ␦ erodimers have a pharmacologic profile distinct from those of the receptors in dorsal root ganglion neurons expressing ␮ receptors ␮ or ␦ homodimers. Because the heterodimers exhibit distinct (15), support the idea that mechanisms and/or proteins that signaling properties, the protein and mechanism regulating their modulate the level of ␮-␦ complexes serve as critical factors in levels have significant effects on morphine-mediated physiology. the development of tolerance (16). Thus, the factors influencing We report the characterization of RTP4, a Golgi chaperone, as a the homodimer-to-heterodimer ratio become a key issue in regulator of the levels of heterodimers at the cell surface. We show determining the net effect of an opiate. Moreover, they could that the association with RTP4 protects ␮-␦ receptors from ubiq- redirect the coupling of opioid receptors to a distinct signal uitination and degradation. This leads to increases in surface transduction pathway (17, 18). But very little is known about the heterodimer levels, thereby affecting signaling.
    [Show full text]
  • Étude Moléculaire De La Formation De Complexes Protéiques Impliqués Dans La Signalisation Des Récepteurs Couplés Aux Protéines G
    Université de Montréal Étude moléculaire de la formation de complexes protéiques impliqués dans la signalisation des récepteurs couplés aux protéines G par Billy Breton Département de Biochimie, Faculté de Médecine Thèse présentée à la Faculté des études supérieures en vue de l’obtention du grade de Ph.D. en Biochimie Mai, 2010 © Billy Breton, 2010 Université de Montréal Faculté des études supérieures Cette thèse intitulée : Étude moléculaire de la formation de complexes protéiques impliqués dans la signalisation des récepteurs couplés aux protéines G présentée par : Billy Breton a été évaluée par un jury composé des personnes suivantes : Sylvie Mader, président-rapporteur Michel Bouvier, directeur de recherche Marc Servant, membre du jury Jean-Philippe Pin, examinateur externe Audrey Claing, représentant du doyen de la FES iii Résumé La communication cellulaire est un phénomène important pour le maintien de l’homéostasie des cellules. Au court des dernières années, cette sphère de recherche sur la signalisation cellulaire a connue des avancées importantes au niveau de l’identification des acteurs principaux impliqués dans la reconnaissance extracellulaire des signaux, ainsi que la compréhension des voies de signalisation engagées par les cellules pour répondre aux facteurs extracellulaires. Malgré ces nouvelles informations, les diverses interrelations moléculaires entre les acteurs ainsi que les voies de signalisation cellulaire, demeurent mal comprises. Le transfert d’énergie de résonance de bioluminescence (BRET) permet la mesure d’interactions protéiques et peut être utilisé dans deux configurations, le BRET480-YFP 1 2 (connu aussi comme le BRET ) et le BRET400-GFP (connu aussi en tant que BRET ). Suite à l’oxydation de son substrat, la luciférase de renilla peut transférer son énergie à une protéine fluorescente, uniquement si elles sont à proximité l’une de l’autre (≤100Å).
    [Show full text]
  • Utilization of Biased G Protein-Coupled Receptor Signaling Towards Development of Safer and Personalized Therapeutics
    molecules Review Utilization of Biased G Protein-Coupled Receptor Signaling towards Development of Safer and Personalized Therapeutics Metehan Ilter 1 , Samman Mansoor 2 and Ozge Sensoy 3,* 1 Department of Biomedical Engineering, The School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul 34810, Turkey; [email protected] 2 Department of Biomedical Engineering and Bioinformatics, The Graduate School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul 34810, Turkey; [email protected] 3 Department of Computer Engineering, The School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul 34810, Turkey * Correspondence: [email protected]; Tel.: +90-(216)-681-5100 (ext. 5621) Academic Editor: Irina S. Moreira Received: 26 April 2019; Accepted: 24 May 2019; Published: 29 May 2019 Abstract: G protein-coupled receptors (GPCRs) are involved in a wide variety of physiological processes. Therefore, approximately 40% of currently prescribed drugs have targeted this receptor family. Discovery of b-arrestin mediated signaling and also separability of G protein and b-arrestin signaling pathways have switched the research focus in the GPCR field towards development of biased ligands, which provide engagement of the receptor with a certain effector, thus enriching a specific signaling pathway. In this review, we summarize possible factors that impact signaling profiles of GPCRs such as oligomerization, drug treatment, disease conditions, genetic background, etc. along with relevant molecules that can be used to modulate signaling properties of GPCRs such as allosteric or bitopic ligands, ions, aptamers and pepducins. Moreover, we also discuss the importance of inclusion of pharmacogenomics and molecular dynamics simulations to achieve a holistic understanding of the relation between genetic background and structure and function of GPCRs and GPCR-related proteins.
    [Show full text]
  • G-Protein-Coupled Receptor Oligomerization and Its Potential for Drug Discovery
    REVIEWS G-PROTEIN-COUPLED RECEPTOR OLIGOMERIZATION AND ITS POTENTIAL FOR DRUG DISCOVERY Susan R. George*‡§, Brian F. O’Dowd*§ and Samuel P. Lee* G-protein-coupled receptors (GPCRs) represent by far the largest class of targets for modern drugs. Virtually all therapeutics that are directed towards GPCRs have been designed using assays that presume that these receptors are monomeric. The recent realization that these receptors form homo-oligomeric and hetero-oligomeric complexes has added a new dimension to rational drug design. However, this important aspect of GPCR biology remains largely unincorporated into schemes to search for new therapeutics. This review provides a synopsis of the current thinking surrounding GPCR homo-oligomerization and hetero- oligomerization and shows how new models point towards unexplored avenues in the development of new therapies. POLYMORPHISM The G-protein-coupled receptors (GPCRs) are the largest assumption that monomeric receptors participate in The occurrence in a population class of cell-surface receptors and are encoded by >1,000 the processes. Despite the fact that, for many other of two or more variant alleles of genes in the human genome1. GPCRs are activated by a classes of receptors, such as the tyrosine-kinase recep- a gene, for which the frequency diverse array of ligands, including hormones, peptides, tors, constitutive or ligand-induced oligomerization has of the rarer alleles is greater than amino acids, ions and photons of light, and transduce long been known to be essential for signalling7, only a can be explained by recurrent mutation alone. signals through a wide range of effectors. Not surpris- monomeric model for GPCRs was generally accepted.
    [Show full text]
  • Cell Surface Targeting of ␮-␦ Opioid Receptor Heterodimers by RTP4
    Cell surface targeting of ␮-␦ opioid receptor heterodimers by RTP4 Fabien M. De´ caillot, Raphael Rozenfeld, Achla Gupta, and Lakshmi A. Devi* Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029 Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved August 22, 2008 (received for review April 29, 2008) ␮ opioid receptors are G protein–coupled receptors that mediate heterodimers, respectively. These findings, together with the the pain-relieving effects of clinically used analgesics, such as observation that the development of tolerance correlates with morphine. Accumulating evidence shows that ␮-␦ opioid het- the enhanced surface expression (through externalization) of ␦ erodimers have a pharmacologic profile distinct from those of the receptors in dorsal root ganglion neurons expressing ␮ receptors ␮ or ␦ homodimers. Because the heterodimers exhibit distinct (15), support the idea that mechanisms and/or proteins that signaling properties, the protein and mechanism regulating their modulate the level of ␮-␦ complexes serve as critical factors in levels have significant effects on morphine-mediated physiology. the development of tolerance (16). Thus, the factors influencing We report the characterization of RTP4, a Golgi chaperone, as a the homodimer-to-heterodimer ratio become a key issue in regulator of the levels of heterodimers at the cell surface. We show determining the net effect of an opiate. Moreover, they could that the association with RTP4 protects ␮-␦ receptors from ubiq- redirect the coupling of opioid receptors to a distinct signal uitination and degradation. This leads to increases in surface transduction pathway (17, 18). But very little is known about the heterodimer levels, thereby affecting signaling.
    [Show full text]
  • Expanding the Concept of G Protein-Coupled Receptor (GPCR) Dimer Asymmetry Towards GPCR-Interacting Proteins Maud Kamal, Pascal Maurice, Ralf Jockers
    Expanding the Concept of G Protein-Coupled Receptor (GPCR) Dimer Asymmetry towards GPCR-Interacting Proteins Maud Kamal, Pascal Maurice, Ralf Jockers To cite this version: Maud Kamal, Pascal Maurice, Ralf Jockers. Expanding the Concept of G Protein-Coupled Receptor (GPCR) Dimer Asymmetry towards GPCR-Interacting Proteins. Pharmaceuticals, MDPI, 2011, 4 (2), pp.273-284. 10.3390/ph4020273. hal-02347809 HAL Id: hal-02347809 https://hal.archives-ouvertes.fr/hal-02347809 Submitted on 5 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Pharmaceuticals 2011, 4, 273-284; doi:10.3390/ph4020273 OPEN ACCESS pharmaceuticals ISSN 1424-8247 www.mdpi.com/journal/pharmaceuticals Review Expanding the Concept of G Protein-Coupled Receptor (GPCR) Dimer Asymmetry towards GPCR-Interacting Proteins Maud Kamal 1,2, Pascal Maurice 1,2 and Ralf Jockers 1,2,* 1 Institut Cochin, Universite Paris Descartes, CNRS, Paris, France 2 Inserm, U1016, Paris, France; E-Mails: [email protected] (M.K.); [email protected] (P.M.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +331-40-51-64-34; Fax: +331-40-51-64-30.
    [Show full text]
  • Oligomerization of the Human Adenosine A2a Receptor Is Driven by 2 the Intrinsically Disordered C-Terminus
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423144; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 OLIGOMERIZATION OF THE HUMAN ADENOSINE A2A RECEPTOR IS DRIVEN BY 2 THE INTRINSICALLY DISORDERED C-TERMINUS 3 Author Line: Khanh D. Q. Nguyen1, Michael Vigers2, Eric Sefah3, Susanna Seppälä2, Jennifer P. 4 Hoover1, Nicole S. Schonenbach2, Blake Mertz3, Michelle A. O’Malley*,2, Songi Han*,1,2. 5 Author Affiliations: 6 1Department of Chemistry and Biochemistry, University of California – Santa Barbara, CA 93106 7 2Department of Chemical Engineering, University of California – Santa Barbara, CA 93106 8 3C. Eugene Bennett Department of Chemistry, West Virginia University, 217 Clark Hall, 9 Morgantown, WV 26506 10 Corresponding Authors: 11 • Songi Han – Santa Barbara, CA 93106; (805) 893-4858; [email protected] 12 • Michelle A. O’Malley – Santa Barbara, CA 93106; (805) 893-4769; 13 [email protected] 14 Classifications: Biological Sciences – Biophysics and Computational Biology 15 Keywords: G protein-coupled receptors, oligomerization, intrinsically disordered protein, C- 16 terminus, depletion interactions, size-exclusion chromatography, molecular dynamics simulations. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423144; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 17 SIGNIFICANCE 18 G protein-coupled receptors (GPCRs) are important drug targets in medicine. While it is widely 19 known that these receptors can form oligomers with unique functional consequences, the driving 20 factor of receptor oligomerization remains unclear.
    [Show full text]
  • Structure and Function of G Protein-Coupled Receptor Oligomers: Implications for Drug Discovery Nicole S
    Advanced Review Structure and function of G protein-coupled receptor oligomers: implications for drug discovery Nicole S. Schonenbach, Sunyia Hussain and Michelle A. O’Malley∗ G protein-coupled receptor (GPCR) oligomers are promising targets for the design of new highly selective therapeutics. GPCRs have historically been attractive drug targets for their role in nearly all cellular processes, and their localization at the cell surface makes them easily accessible to most small molecule therapeutics. However, GPCRs have traditionally been considered a monomeric entity, a notion that greatly oversimplifies their function. As evidence accumulates that GPCRs tune function through oligomer formation and protein–protein interactions, we see a greater demand for structural information about these oligomers to facilitate oligomer-specific drug design. These efforts are slowed by difficulties inherent to studying membrane proteins, such as low expression yield, in vitro stability and activity. Such obstacles are amplified for the study of specific oligomers, as there are limited tools to directly isolate and characterize these receptor complexes. Thus, there is a need to develop new interdisciplinary approaches, combining biochemical and biophysical techniques, to address these challenges and elucidate structural details about the oligomer and ligand binding interfaces. In this review, we provide an overview of mechanistic models that have been proposed to underlie the function of GPCR oligomers, and perspectives on emerging techniques to characterize GPCR oligomers for structure-based drug design. © 2014 Wiley Periodicals, Inc. Howtocitethisarticle: WIREs Nanomed Nanobiotechnol 2014. doi: 10.1002/wnan.1319 INTRODUCTION contain an , ,and subunit, and are linked to several different signaling cascades that have been protein-coupled receptors (GPCRs) are an most studied for Class A GPCRs.
    [Show full text]
  • Downloaded from Bioscientifica.Com at 10/02/2021 07:36:32AM Via Free Access
    57:1 G KLEINAU and others GPCR oligomerization 57:1 R59–R80 Review Oligomerization of GPCRs involved in endocrine regulation Correspondence Gunnar Kleinau, Anne Müller and Heike Biebermann should be addressed to H Biebermann Institute of Experimental Pediatric Endocrinology (IEPE), Charité-Universitätsmedizin, Berlin, Germany Email [email protected] Abstract More than 800 different human membrane-spanning G-protein-coupled receptors Key Words (GPCRs) serve as signal transducers at biological barriers. These receptors are activated by f G-protein-coupled a wide variety of ligands such as peptides, ions and hormones, and are able to activate receptors a diverse set of intracellular signaling pathways. GPCRs are of central importance in f homomers endocrine regulation, which underpins the significance of comprehensively studying f heteromers these receptors and interrelated systems. During the last decade, the capacity for f homooligomerization multimerization of GPCRs was found to be a common and functionally relevant property. f heterooligomerization The interaction between GPCR monomers results in higher order complexes such as f dimers homomers (identical receptor subtype) or heteromers (different receptor subtypes), f oligomers which may be present in a specific and dynamic monomer/oligomer equilibrium. It is f endocrine regulation widely accepted that the oligomerization of GPCRs is a mechanism for determining f endocrine diseases the fine-tuning and expansion of cellular processes by modification of ligand action, f signaling expression levels, and related signaling outcome. Accordingly, oligomerization provides exciting opportunities to optimize pharmacological treatment with respect to receptor Journal of Molecular Endocrinology target and tissue selectivity or for the development of diagnostic tools. On the other hand, GPCR heteromerization may be a potential reason for the undesired side effects of pharmacological interventions, faced with numerous and common mutual signaling modifications in heteromeric constellations.
    [Show full text]
  • G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives
    1521-0081/66/2/413–434$25.00 http://dx.doi.org/10.1124/pr.113.008052 PHARMACOLOGICAL REVIEWS Pharmacol Rev 66:413–434, April 2014 U.S. Government work not protected by U.S. copyright ASSOCIATE EDITOR: MARK P. MATTSON G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives Sergi Ferré, Vicent Casadó, Lakshmi A. Devi, Marta Filizola, Ralf Jockers, Martin J. Lohse, Graeme Milligan, Jean-Philippe Pin, and Xavier Guitart Integrative Neurobiology Section, National Institute on Drug Addiction, Intramural Research Program, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland (S.F., X.G.); Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain (V.C.); Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Barcelona, Spain (V.C.); Department of Pharmacology and Systems Therapeutics (L.A.D.) and Department of Structural and Chemical Biology (M.F.), Icahn School of Medicine at Mount Sinai, Mount Sinai, New York; Institut National de la Santé et de la Recherche Médicale, U1016, Institut Cochin, Paris, France (R.J.); Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Paris, France (R.J.); Universite Paris Descartes, Sorbone Paris Cite, Paris, France (R.J.); Institute of Pharmacology and Toxicology and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany (M.J.L.); Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (G.M.); Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Université Montpellier, Montpellier, France (J.-P.P.); and Institut National de la Santé et de la Recherche Médicale, U661, Montpellier, France (J.-P.P.) Downloaded from Abstract.
    [Show full text]
  • Oligomerisation of G-Protein-Coupled Receptors
    COMMENTARY Signal Transduction and Cellular Organization 1265 Oligomerisation of G-protein-coupled receptors Graeme Milligan Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK ([email protected]) Journal of Cell Science 114, 1265-1271 © The Company of Biologists Ltd Summary A range of approaches have recently provided evidence effective delivery of newly synthesised receptors to the cell that G-protein-coupled receptors can exist as oligomeric surface is a developing concept, and recent studies have complexes. Both homo-oligomers, comprising multiple employed a series of energy-transfer techniques to explore copies of the same gene product, and hetero-oligomers the presence and regulation of receptor oligomerisation in containing more than one receptor have been detected. In living cells. However, the majority of studies have relied several, but not all, examples, the extent of oligomerisation largely on co-immunoprecipitation techniques, and there is is regulated by the presence of agonist ligands, and still little direct information on the fraction of receptors emerging evidence indicates that receptor hetero-oligomers existing as oligomers in intact cells. can display distinct pharmacological characteristics. A chaperonin-like role for receptor oligomerisation in Key words: Receptor, G protein, Signal transduction Introduction within the cell. However, the recognition that GPCRs can exist The family of G-protein-coupled receptors (GPCRs) is one of as dimers or higher oligomers has recently initiated some of the largest in the human genome. They act as the recognition the most fevered debate in the area. elements for a vast range of hormones, transmitters and modulators (Bockaert and Pin, 1999).
    [Show full text]