Methods in cell biology, volume 132

Methods in Cell Biology G Protein-Coupled Receptors: Signaling, Trafficking and Regulation Volume 132 Series Editors Leslie Wilson Department of Molecular, Cellular and Developmental Biology University of California Santa Barbara, California Phong Tran University of Pennsylvania Philadelphia, USA & Institut Curie, Paris, France Methods in Cell Biology G Protein-Coupled Receptors: Signaling, Trafficking and Regulation Volume 132 Edited by Arun K Shukla Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 125 London Wall, London EC2Y 5AS, UK The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2016 Copyright © 2016 Elsevier Inc All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein) Notices Knowledge and best practice in this field are constantly changing As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein ISBN: 978-0-12-803595-5 ISSN: 0091-679X For information on all Academic Press publications visit our website at http://store.elsevier.com Contributors Agnes M Acevedo Canabal Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA D Agranovich Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Stefan Amisten Diabetes Research Group, King’s College London, London, UK Gabriela Antunes Laboratory of Neural Systems (SisNE), Department of Physics, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, Brazil Chaitanya A Athale Division of Biology, IISER Pune, Pune, India Nicolas Audet Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada Mohammed Akli Ayoub Biologie et Bioinformatique des Syste`mes de Signalisation, Institut National de la Recherche Agronomique, UMR85, Unite´ Physiologie de la Reproduction et des Comportements; CNRS, UMR7247, Nouzilly, France; LE STUDIUMÒ Loire Valley Institute for Advanced Studies, Orle´ans, France R Bar-Shavit Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Damian Bartuzi Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, Lublin, Poland Maik Behrens Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany xiii xiv Contributors Nicolas F Berbari Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA He´le`ne Bonin Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada Michel Bouvier Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada Amitabha Chattopadhyay CSIR-Center of Cellular and Molecular Biology, Hyderabad, India Linjie Chen Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China Santiago Cuevas Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington University School of Medicine and Health Sciences, WA, USA Francheska Delgado-Peraza Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA Dominic Devost Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC, Canada Antonella Di Pizio Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, Israel Shalini Dogra Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Zyanya P Espinosa-Riquer Departamento de Farmacobiologı´a, Centro de Investigacio´n y de Estudios Avanzados del IPN, Me´xico D.F., Mexico Timothy N Feinstein Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Contributors Colleen A Flanagan School of Physiology and Medical Research Council Receptor Biology Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Wits Parktown, Johannesburg, South Africa Alexandre Gidon Molecular Mechanisms of Mycobacterial Infection, Center for Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway Claudia Gonza´lez-Espinosa Departamento de Farmacobiologı´a, Centro de Investigacio´n y de Estudios Avanzados del IPN, Me´xico D.F., Mexico S Grisaru-Granovsky Department of Obstetrics and Gynecology, Shaare Zedek, Jerusalem, Israel Aylin C Hanyaloglu Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Terence E He´bert Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC, Canada Mellisa M Hege Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA Ilpo Huhtaniemi Institute of Reproductive and Developmental Biology, Imperial College London, London, UK M Jaber Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Kim C Jonas Institute of Reproductive and Developmental Biology, Imperial College London, London, UK; Institute of Medical and Biomedical Education, St George’s University of London, London, UK Pedro A Jose Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington University School of Medicine and Health Sciences, WA, USA Manali Joshi Savitribai Phule Pune University, Pune, India xv xvi Contributors Agnieszka A Kaczor Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, Lublin, Poland; School of Pharmacy, University of Eastern Finland, Kuopio, Finland A Kancharla Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Rafik Karaman Bioorganic Chemistry Department, Faculty of Pharmacy, Al-Quds University, Jerusalem, Israel Hiroyuki Kobayashi Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada Ajeet Kumar Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Christian Le Gouill Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada Anat Levit Department of Pharmaceutical Chemistry, University of California e San Francisco, San Francisco, CA, USA Bin Lu Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China Viktorya Lukashova Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada Marina Macı´as-Silva Departamento de Biologı´a Celular y Desarrollo, Instituto de Fisiologı´a Celular, Universidad Nacional Auto´noma de Me´xico, Me´xico D.F., Mexico M Maoz Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Contributors Dariusz Matosiuk Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, Lublin, Poland Jeremy C McIntyre Department of Neuroscience, University of Florida, Gainesville, FL, USA; Center for Smell and Taste, University of Florida, Gainesville, FL, USA Masha Y Niv Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, Israel; Fritz Haber Center for Molecular Dynamics, The Hebrew University, Jerusalem, Israel Carlos Nogueras-Ortiz Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA Melanie Philipp Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany Cristina Roman-Vendrell Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Physiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA Ewelina Rutkowska Department of Biopharmacy, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, Lublin, Poland Jana Selent Research Programme on Biomedical Informatics (GRIB), Universitat Pompeu Fabra, IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain Durba Sengupta CSIR-National Chemical Laboratory, Pune, India Ying Shi Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China Fabio Marques Simoes de Souza Center for Mathematics, Computation and Cognition, Federal University of ABC, Sa˜o Bernardo Campo, Brazil xvii xviii Contributors Michal Slutzki Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, Israel Chandan Sona Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Katarzyna M Targowska-Duda Department of Biopharmacy, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, Lublin, Poland Teresa Casar Tena Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany B Uziely Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Genaro Va´zquez-Victorio Departamento de Biologı´a Celular y Desarrollo, Instituto de Fisiologı´a Celular, Universidad Nacional Auto´noma de Me´xico, Me´xico D.F., Mexico Jean-Pierre Vilardaga Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Van Anthony M Villar Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington University School of Medicine and Health Sciences, WA, USA Richard Wargachuk Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC, Canada Kunhong Xiao Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Prem N Yadav Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Guillermo A Yudowski Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA Volumes in Series Volume 128 (2015) Building a Cell from its Component Parts Edited by Jennifer Ross and Wallace F Marshall Volume 129 (2015) Centrosome & Centriole Edited by Renata Basto and Karen Oegema Volume 130 (2015) Sorting and Recycling Endosomes Edited by Wei Guo Volume 131 (2016) The Neuronal Cytoskeleton, Motor Proteins, and Organelle Trafficking in the Axon Edited by K Kevin Pfister 465 Index Note: Page numbers followed by “f ” indicate figures, “t” indicate tables and “b” indicates boxes A A-kinase-anchoring proteins (AKAP), 169 A2AR, see Adenosine 2A receptor (A2AR) AA, see Arachidonic acid (AA) Ab-initio approaches, 433 ABPs, see Actin-binding proteins (ABPs) AC, see Adenylate cyclase (AC) AC3, see Adenylyl cyclase III (AC3) ACE2, see Angiotensin-converting enzyme (ACE2) Acetonitrile (ACN), 124 Acidic region (AD), 348 ACN, see Acetonitrile (ACN) Actin, 169e170 polymerization GPCR/G12/13/RHO axis and, 171e172 GPCR/GS/AC/CAMP axis and, 172e173 staining protocol with anti-actin antibody, 183e184 Actin-associated proteins, see Actin-binding proteins (ABPs) Actin-binding proteins (ABPs), 166e167, 169e170 Actinecytoskeleton dynamics, 169e171, 170f rearrangements, 180f cellular processes controlled by, 173e177 protocols to evaluating, 177e178 AD, see Acidic region (AD) Adaptive PoissoneBoltzmann Solver (APBS), 384 Adenosine 2A receptor (A2AR), 415 Adenosine triphosphate (ATP), 168e169 Adenylate cyclase (AC), 168e169 Adenylyl cyclase III (AC3), 132 Adherent cells, 14e15 Adhesion, 174 Adipokinetic hormone (AKH), 306 Adipokinetic hormone receptor (AKHR), 296, 306 coding sequence, 302 Adipose tissue biopsies handling materials, 74e75 methods, 76 GPCR mRNA expression screening, 82e85 homogenization materials, 75 methods, 76e77 RNA purification and cDNA generation materials, 75 methods, 77e81 ADRA2A, 86te96t Adrenergic receptor (AR), 362e363 AEA, see Anandamide (AEA) 2-AG, see 2-araquidonoylglycerol (2-AG) Agonists, 296e297, 307e308, 410e414 antagonists vs., 414e420 AKAP, see A-kinase-anchoring proteins (AKAP) AKH, see Adipokinetic hormone (AKH) AKHR, see Adipokinetic hormone receptor (AKHR) Allosteric Database (ASD), 377 Allosterism, 376, see also Biased signaling; Dimerization; Ligandereceptor interactions finding allosteric site, 377e378 intricate mechanism investigation, 378e380 a-MCD, see Methyl-a-cyclodextrin (a-MCD) AMBER, 378e379 American Type Culture Collection (ATCC), 300 Amphipathic lipids, Anandamide (AEA), 175 Anaphylactic degranulation, 175e176 Angiotensin II type (AT1R), 266e269 Angiotensin-converting enzyme (ACE2), 342e343 Antagonists, 414e420 Antisense MOs, 150 delivery of, 151e153 APBS, see Adaptive PoissoneBoltzmann Solver (APBS) Apoptosis, 176 AR, see Adrenergic receptor (AR) Arachidonic acid (AA), 375e376 2-araquidonoylglycerol (2-AG), 175 Arginine vasopressin-induced sensor (AVPinduced sensor), 327f Arrestins, 5e6, 234e236 ASD, see Allosteric Database (ASD) Association rate constant (kon), 193 AT1R, see Angiotensin II type (AT1R) ATCC, see American Type Culture Collection (ATCC) Atomistic molecular dynamic simulations, 442 467 468 Index ATP, see Adenosine triphosphate (ATP) AutoDock, 365e367 AVP-induced sensor, see Arginine vasopressininduced sensor (AVP-induced sensor) B BallesteroseWeinstein numbering method (BW numbering method), 406e407 BardeteBiedl syndrome (BBS), 36e37 b-adrenergic receptor kinase (b-ARK), 346e347 b-arrestin, 5e6, 167e168, 295e297 b-arrestin1, 110e112 b-arrestin2, 46, 110e112, 296 recruitment, 266e269 assays, 236e239, 238f b-blockers, 218e219 b-MCD, see Methyl-b-cyclodextrin (b-MCD) b1-adrenergic receptor (b1ADR), 415 b2-adrenergic receptor (b2ADR), 218, 407, 441f Biased agonism, 234e236, 320e321 Biased agonists, 373 Biased signaling, 218e219, see also Allosterism; Dimerization; Ligandereceptor interactions building and simulating conformational receptor states, 373e375 modeling biased agonism importance, 372e373 rational design of biased molecules, 375e376 structural information, 373 transforming balanced natural agonist serotonin, 376f Binding equilibrium, 201, 205e206, 208e209 Bio-Rad protein assay, 220e221 Bioluminescence resonance energy transfer (BRET), 16e18, 237, 256, 295, 321e322, 324f Biosensors, 320e321 Bitter taste, 403 BorneOppenheimer approximation, 435 Bovine serum albumin (BSA), 13, 179 BRET, see Bioluminescence resonance energy transfer (BRET) BRET-based ERK-NLS sensor nuclear ERK activation measurement in endogenous cell model experimental workflow, 332 results, 332e334 theory, 330e332 nuclear ERK activation measurement in heterologous cell models experimental workflow, 328 results, 328e330 theory, 328 BSA, see Bovine serum albumin (BSA) Buffer, 207 BW numbering method, see BallesteroseWeinstein numbering method (BW numbering method) C C-laurdan, 12 C-terminal domain of Renilla luciferase (RlucC), 302 C-terminal fragment of luciferase (DnaEC-lucC), 297 C-X-C chemokine receptor type (CXCR4), 415 C5aR, see Complement 5a receptor (C5aR) Ca2+-activated chloride channels (CAC channels), 132 Caenorhabditis elegans (C elegans), 42e43 Calponin homology domain (CH domain), 348 cAMP, see Cyclic adenosine monophosphate (cAMP) Cannabinoid receptors, 175 Canonical binding site, 410e413 Carvedilol, 218e219 Caveolae, 4e5, 10 Caveolin-1, 10 CC chemokine receptor (CCR3), 369e370 CCK2, see Cholecystokinin (CCK2) CCR3, see CC chemokine receptor (CCR3) CCR7 receptor, 174 Cdc42, 171 cDNA, see complementary DNA (cDNA) cdt, see Cytolethal distending toxin (cdt) Cell cell-based assay, 239e240, 295, 312 culture, 7e8 GPCR organization in cell membrane, 446e447 line revival and maintenance, 242 lysis, 220e222 motility, 174 pellet collection, 7e8 in suspension, 13e14 trypsinization, 242e243 CG MD, see Coarse-grained MD (CG MD) CH domain, see Calponin homology domain (CH domain) CHARMM, 368, 378e379 CHARMM-GUI platform, 373e376 Chemokines, 174 Chemosensory, 403 Chirp Z-transform (CZT), 377 Index Cholecystokinin (CCK2), 272e274 Cholera toxin subunit B (CTxB), 10 labeling, 11e12 Cholesterol, Cholesterol-dependent dimer, 446 Cilia GPCR, 37 dynamic trafficking, 45e47 olfactory sensory neuron, 40f, 41e45 in other neurons, 39e41 photoreceptor, 38f rhodopsin as ciliary GPCR, 38e39 Ciliopathy, 37 Cilium, 36e37 cJun N-terminal kinase (JNK), 321 Class A GPCRs, 403 to bitter taste receptors comparison, 402 ligandereceptor interactions, 410e420 sequence alignments and GPCR signature motifs, 404e410, 405t CNG channels, see Cyclic nucleotide gated channels (CNG channels) Coarse-grain model, 435 simulations, 443e445 Coarse-grained MD (CG MD), 361 Coimmunoprecipitation, 122e123 Competition binding assays, see Indirect binding assays Complement 5a receptor (C5aR), 258e259 complementary DNA (cDNA), 302 CoMSIA, 370e371 Confocal FRET, 113 microscopic system, 113 Conformation-based assays, 237 Conformational receptor states, 373e375 Continuum electrostatic models, 436 Cp, see Crossing point (Cp) Crossing point (Cp), 159 CTxB, see Cholera toxin subunit B (CTxB) CXCR4, see C-X-C chemokine receptor type (CXCR4) Cyclic adenosine monophosphate (cAMP), 114e116, 132, 168e169, 265e266 Cyclic nucleotide gated channels (CNG channels), 132 Cytolethal distending toxin (cdt), 11e12 Cytoskeleton, 169e170 CZT, see Chirp Z-transform (CZT) D D1 dopamine receptor (D1R), 11f D1R, see D1 dopamine receptor (D1R) D3R, see Dopamine D3 receptor (DRD3) DAG, see Diacylglycerol (DAG) DAPI, see 40 ,6-diamidino-2-phenylindole (DAPI) Data analysis, 245e247 for agonist activity, 245e246 plotting normalized agonist response, 246 plotting RLU, 246 for antagonistic activity, 246e247 plotting normalized antagonistic response, 247 plotting RLU, 247 Data interpretation, 10 Dbl homology (DH), 347e348 DCT, see Discrete cosine transform (DCT) DDM, see n-Dodecyl b-D-maltoside (DDM) DeepBlueC, 237 DBRET2 ratio, 325 d-opioid receptor (OPRD), 415 Density functional theory, 433 Depletion, 204 Desensitization, Desert Hedgehog (Dhh), 148 Detergent-based method, Detergent-free method, 7e9 Deterministic models, 436e437 DFT, see Fourier transform (DFT) DH, see Dbl homology (DH) Dhh, see Desert Hedgehog (Dhh) Diacylglycerol (DAG), 169 40 ,6-diamidino-2-phenylindole (DAPI), 13 Dilution of test compounds for assay, 243e244 Dimerization, 56e57, 62e63, 68, 130, 380, see also Allosterism; Biased signaling; Ligandereceptor interactions ligand design for GPCR oligomers, 384e385 sequence-based methods, 381 structure-based methods, 381e384 Discrete cosine transform (DCT), 377 Dishevelled (DVL), 352 Displacement binding assays, see Indirect binding assays Disulfide bonds, 362e363 DMEM, see Dulbecco’s Modified Eagle Medium (DMEM) DnaE intein, 296e297 DnaEC-lucC, see C-terminal fragment of luciferase (DnaEC-lucC) DOCK, 365e366 Docking score, 439 Docking studies, 438 6-dodecanoyl-2-(dimethylamino)-naphthalene, see Laurdan 469 470 Index 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl) amino]-naphthalene), see C-laurdan Dopamine, 249e250 Dopamine D3 receptor (DRD3), 362e363, 415 Dopamine D4 receptor (DRD4), 249e250 Dopamine receptor (DRD1), 45e46 DPBS, see Dulbecco’s phosphate-buffered saline (DPBS) DRD3, see Dopamine D3 receptor (DRD3) Drug plate preparation and drug addition, 243e245 compound addition and plate incubation agonist assay, 244e245 antagonist assay, 245 dilution of test compounds for assay, 243e244 substrate addition and luminescence measurement, 245 Dual thrombin receptor system, 344 Dulbecco’s Modified Eagle Medium (DMEM), 178e179, 304, 306 Dulbecco’s phosphate-buffered saline (DPBS), 179 DVL, see Dishevelled (DVL) E Early endosomes, 5e6 isolation, 121 EC environment, see Extracellular environment (EC environment) ECD, see Extracellular N-terminal domain (ECD) ECLs, see Extracellular loops (ECLs) ECM, see Extracellular matrix (ECM) EFC, see Enzyme fragment complementation (EFC) EKAR sensor, see Extracellular-regulated kinase activity reporter sensor (EKAR sensor) Elastic network model (ENM), 380 Electron multiplying charged-coupled device camera (EM-CCD), 64e65 Electrostatics studies, 383e384 EM-CCD, see Electron multiplying chargedcoupled device camera (EM-CCD) Endocytic events, 30e31 Endocytosis, 29e31 Endosomal cAMP production, studying regulation of, 110e112 experimental procedures, 114e124 materials confocal FRET, 113 FRET analysis, 113e114 FRET principle, 112 reagents, 112 TIRF FRET, 113 wide-field FRET, 112e113 Endosomal colocalization analysis, 119e121 Endosomal domain trafficking of PTHR, 119e121 endosomal colocalization analysis, 119e121 image trafficking of PTHR-GFP, 119, 120f transfect HEK293 cells, 119 Endosomal GPCR proteomics, 121e124 coimmunoprecipitation, 122e123 early endosome isolation, 121 in-solution digestion and peptide desalting, 123e124 Endosomal PH, 118 analyzing pH variation, 118 measuring pH variation along endocytic pathway of PTHR, 118 standard curve for pH, 118 Endosomal PTH/PTHR localization, 116e118 Endosomal receptor dynamics, 116 Endothelin, 176 ENM, see Elastic network model (ENM) Enzyme complementation assays, 237e239 Enzyme fragment complementation (EFC), 295 Enzyme-mediated activation of radical sources, 12 Equilibrium affinity constant (Ka), 193 ER, see Estrogen receptor (ER) ERK biosensor, 323 ERK-NLS sensor, 326 ERK1/2, see Extracellular signal-regulated kinase and (ERK1/2) ERM proteins, see Ezrin/radixin/moesin proteins (ERM proteins) Estrogen receptor (ER), 342 Etk/Bmx, 345e349 Eukaryotic cells, 167 Eukaryotic cytoskeleton, 166 EVT, see Extravillous trophoblast (EVT) Exocytosis, see Secretion Exocytotic events, 30 Extracellular environment (EC environment), 402 Extracellular loops (ECLs), 404 ECL2, 362e363, 366e367 Extracellular matrix (ECM), 172 Extracellular N-terminal domain (ECD), 66e67 Extracellular signal-regulated kinase and (ERK1/2), 265e266, 321 signaling, 345 Extracellular-regulated kinase activity reporter sensor (EKAR sensor), 321e322 Extravillous trophoblast (EVT), 346 Ezrin/radixin/moesin proteins (ERM proteins), 172 Index F F-actin, see Filamentous/polymeric-actin (F-actin) Fast odorant adaptation, 133 FBS, see Fetal Bovine Serum (FBS) FCS, see Fetal calf serum (FCS); Fluorescence correlation spectroscopy (FCS) Fetal Bovine Serum (FBS), 178e179 Fetal calf serum (FCS), 63 FFA1, see Free fatty acid receptor1 (FFA1) Filamentous/polymeric-actin (F-actin), 169e170 Filamin, 173 Firefly luciferase, 303 Firefly luciferase assay, 308e311 FLAG immunoprecipitation, 222 FlexX, 365e366 FLIM-FRET, see Fluorescence lifetime imaging microscopyefluorescence resonance energy transfer (FLIM-FRET) FLOG, 365e366 Flotillin-1, 10 Fluorescence correlation spectroscopy (FCS), 57 Fluorescence energy resonance transfer (FRET), 237, 256, 295 Fluorescence lifetime imaging microscopye fluorescence resonance energy transfer (FLIM-FRET), 12e13 fMet-Leu-Phe (fMLP), 174 Follicle-stimulating hormone (FSHR), 266e269 Force field, 435 Forskolin, 173 Foărster resonance energy transfer (FRET), 112 analysis, 113e114 principle, 112 Foărsters law, 257e258 Fourier transform (DFT), 377 Free fatty acid receptor1 (FFA1), 247e248 FRET, see Fluorescence energy resonance transfer (FRET); Foărster resonance energy transfer (FRET) Frustratometer, 380 FSHR, see Follicle-stimulating hormone (FSHR) Functional selectivity, 218e219, 320e321 Fusion expression constructs, 300e303 plasmid vectors construction for assay system GPCReRab5 interaction, 302e303 GPCReb-arrestin interaction, 302 G G protein-coupled receptor 161 (GPR161), 342 G protein-coupled receptor kinases (GRKs), 167e168, 237e239, 259e261, 295 GRK3, 133e134 G protein-coupled receptors (GPCRs), 5e6, 26e27, 36e37, 42f, 56, 74, 127e128, 130, 148, 166e169, 192, 218, 234, 256, 295, 320e321, 342, 360e361, 430e431, 430f, see also Cilia GPCR; Lipid rafts; Protease-activated receptors (PARs) actinecytoskeleton dynamics modulated by, 178e179 allosterism, 376 finding allosteric site, 377e378 intricate mechanism investigation, 378e380 analyzing GPCReligand interactions dynamics, 437 binding of carazalol to b2-adrenergic receptor, 439f ligands classification, 439 molecular basis of population-wide variations, 440e441 natural ligands and drugs, 438e439 pathways of ligand entry and exit, 440 unravel biology of senses, 437e438 biased signaling building and simulating conformational receptor states, 373e375 modeling biased agonism importance, 372e373 rational design of biased molecules, 375e376 structural information, 373 transforming balanced natural agonist serotonin, 376f biosynthesis and degradation, 219 cell lysis, 220e222 discussion, 227e230 expressing FLAG-b2AR, 223e224 heterologous FLAG-b2AR expression, 228fe229f immunoprecipitation, 222 induction conditions, 220 pulse of FLAG-b2AR expression, 224e225 reagents, 219e220 results in altered signaling profiles, 226e227 signaling assays, 222e223 tissue culture, 220 tracking life cycle of b2AR, 225e226 western blotting, 220e222 workflow of experiments, 223 cellular processes controlled by actinecytoskeleton rearrangements, 173e177 dimerization, 380 ligand design for GPCR oligomers, 384e385 sequence-based methods, 381 471 472 Index G protein-coupled receptors (GPCRs) (Continued) structure-based methods, 381e384 dimerization and oligomerization, 56e57 dynamic interplay between membrane and receptors, 443 GPCR association, 445e446 with membrane, 444fe445f membrane lipid interactions, 443e445 expression quantification, 97e98 functional dynamics, 431 GPCR/G12/13/RHO axis, 171e172 GPCR/GS/AC/CAMP axis, 172e173 GPCReG protein interaction, 261e265, 263f imaging methodologies to single GPCR molecules, 57 principles of dual color PD-PALM, 60f super-resolution imaging of single molecules, 59e61 visualizing individual GPCRS, 58 ligandereceptor interactions materials, 74e76 MD simulations, 361 milestones, 367e370 methods, 76e100 multiscale simulations, 432e437 organization in cell membrane, 446e447 phospholipid bilayer, 434f prediction of receptor structure, 362e364 signaling, 135 and trafficking, 5e6 signature motifs, 404e410 simulation techniques to GPCR activation, 441e442 analyzing signaling complex formation, 442 inactive to active state transitions, 442 steps of homology modeling, 363e364 VS in GPCR ligand search, 371e372 molecular docking, 364e367 SAR in ligand recognition, 370e371 G proteins, 167e169 G-actin, see Globular/monomeric-actin (G-actin) G12 family, 169 G418, 305 GDP, see Guanosine diphosphate (GDP) GEF, see Guanine nucleotide exchange factor (GEF) Generalized Born methods, 436 Gillespie models, 436e437 Gli transcription factors, 148 Glide, 365e367 Globular/monomeric-actin (G-actin), 169e170 Glucagon-like-1 (GLP-1R), 266e269 Glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin system (GRAFS system), 402e403 Glycosylphosphatidylinositol-linked proteins (GPI-linked proteins), GNBPs, see Guanine nucleotide-binding proteins (GNBPs) GnRH, see Gonadotropin-releasing hormone (GnRH) GOLD, 365e367 Gonadotropin-releasing hormone (GnRH), 39e40 Google’s Exacycle cloud-computing, 442 GPCR-radioligand binding assays, 192 calculations, 210e212 components of binding assays, 202 buffer, 207 incubation, 208 radioligand, 204e207 receptor preparation, 202e204 receptor-bound radioligand separation, 208e210 types of binding assays, 192 equilibrium affinity constant, 193 indirect binding assays, 197e200 kinetic binding assays, 200e201 NSB, 193e194 saturation binding assays, 195e196 GPCR/G protein signaling and actin polymerization, 172f GPCR/G12/13/RHO axis and actin polymerization, 171e172 GPCR/GS/AC/CAMP axis and actin polymerization, 172e173 GPCReRab5 interaction, 302e303 GPCRs, see G protein-coupled receptors (GPCRs) GPCReb-arrestin interaction, 302 GPI-linked proteins, see Glycosylphosphatidylinositol-linked proteins (GPI-linked proteins) GPR109A internalization, 296e297 GPR161, see G protein-coupled receptor 161 (GPR161) Gq/11 family, 169 GRAFS system, see Glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin system (GRAFS system) GRAMM-X, 381e382 GRK2, 148 GRKs, see G protein-coupled receptor kinases (GRKs) GROMACS, 368, 378e379 Gs protein family, 168e169 Index GTP, see Guanidine triphosphate (GTP); Guanosine triphosphate (GTP) GTPase, 168 Guanidine triphosphate (GTP), 294 Guanine nucleotide exchange factor (GEF), 168e169, 348 Guanine nucleotide protein-coupled receptors, see G protein-coupled receptors (GPCRs) Guanine nucleotide-binding proteins (GNBPs), 347e348 Guanosine diphosphate (GDP), 127e128 Guanosine triphosphate (GTP), 127e128 GW9508, 247e248, 248f H HA, see Hemagglutinin (HA) HartreeeFock methods, 433 HB, see Homogenization buffer (HB) hCG, see human chorionic gonadotropin (hCG) Heat shock protein 90 (HSP90), 169 Hedgehog signaling (Hh signaling), 148e149, 149t manipulation in zebrafish, 150e153 antisense MOs, 150 delivery of antisense MOs, 151e153 injection setup for zebrafish embryos, 152f smoothened inhibition by small chemical compounds, 153 Hedgehog-dependent organ development, 149e150 smoothened-mediated HH signaling analysis in zebrafish, 150e160 HEK, see Human embryonic kidney (HEK) HEK 293 cells generation, 303e306 HEK 293 cells cotransfection, 304 selection, 304e306 Hemagglutinin (HA), 258e259 Hepatic C9 cell line, 178e179 Heterologous cell systems, 46e47 Heterotrimeric G proteins, 168e169, 256 Hh signaling, see Hedgehog signaling (Hh signaling) High-resolution X-ray structures, 407 High-throughput screening (HTS), 257e258, 371e372 Highest occupied MO (HOMO), 433 Histamine H1 receptor (HRH1), 415 hM3R, see Human muscarinic acetylcholine receptor (hM3R) Homeostatic hippo, 177 HOMO, see Highest occupied MO (HOMO) Homogeneous time-resolved fluorescence (HTRF), 257e258 Homogenization buffer (HB), 121 Homology modeling of GPCRs, 363e364 Horse serum (HS), 179 HRH1, see Histamine H1 receptor (HRH1) HS, see Horse serum (HS) HSP90, see Heat shock protein 90 (HSP90) 5HT1B, see Serotonin receptor 1B (5HT1B) 5HT2A receptor (5HT2AR), 375 5HT2B activation, 418e419 HTLA cell line, transient transfection in, 243 HTRF, see Homogeneous time-resolved fluorescence (HTRF) HTS, see High-throughput screening (HTS) human chorionic gonadotropin (hCG), 62 Human embryonic kidney (HEK), 10 Human muscarinic acetylcholine receptor (hM3R), 369e370 Human placenta, PARs and, 350e352 Hydrogen bond network, 407 Hydrophobic sandwich, 365 I IBMX, see 3-isobutyl-1-methylxanthine (IBMX) IC modules, see Intracellular modules (IC modules) ICLs, see Intracellular loops (ICLs) IF, see Immunofluorescence (IF) IFD, see Induced fit docking (IFD) IFT, see Intraflagellar transport (IFT) Ihh, see Indian Hedgehog (Ihh) Image analysis, 30e31 Imaging methodologies to single GPCR molecules, 57 principles of dual color PD-PALM, 60f super-resolution imaging of single molecules, 59e61 visualizing individual GPCRS, 58 Immunoblotting, 10 Immunofluorescence (IF), 177e178 protocols for, 179e184 Immunoprecipitation, 222 In-solution digestion, 123e124 Incubation, 208 Indian Hedgehog (Ihh), 148 Indirect binding assays, 197e200 Induced fit docking (IFD), 366 Induction conditions, 220 Inositol 1, 5-trisphosphate (IP3), 4, 169 Inositol phosphate (IP), 265e266, 375e376 Inteins, 295e296 473 474 Index Intracellular loops (ICLs), 404 Intracellular modules (IC modules), 404 Intraflagellar transport (IFT), 36e37 Intricate mechanism investigation, 378e380 “Ionic lock” mechanism, 406e407 IP, see Inositol phosphate (IP) IP3, see Inositol 1,4,5-trisphosphate (IP3) 3-isobutyl-1-methylxanthine (IBMX), 173 J JNK, see cJun N-terminal kinase (JNK) JSUBST matrices, see MP-specific substitution matrices (JSUBST matrices) K k-opioid receptor (OPRK), 369, 415 kOR, see k-opioid receptor (OPRK) Kif3a, 148 Kinetic binding assays, 200e201 Kinetic Monte Carlo methods, 436e437 L L-R signaling, see Ligandereceptor signaling (L-R signaling) Langmuir adsorption isotherm, 195e196 LARG, see Leukemia-associated RhoGEF (LARG) Laurdan, 12 Leukemia-associated RhoGEF (LARG), 171 LHR, see Luteinizing hormone receptor (LHR) LibDock, 365e366 LifeAct, 171 Lifeact-mTurquoise2-based protocol for actin filaments staining, 183 Ligand binding, 258e259 classification, 439 depletion, 194, 203, 210e211 design for GPCR oligomers, 384e385 Ligand-induced conformational changes, 259e261, 261f Ligandereceptor interactions, 410e420, 416f, see also Allosterism; Biased signaling; Dimerization agonists, 410e414 antagonists vs agonists, 414e420, 417fe418f vs in GPCR ligand search, 371e372 molecular docking, 364e367 positions involved in ligand binding in TAS2Rs and class A GPCRs, 411te413t SAR in ligand recognition, 370e371 Ligandereceptor signaling (L-R signaling), 110e112 Lipid rafts, fraction preparation, 8e9 GPCR signaling in, 15 changing cholesterol content, 16 fluorescence imaging, 16e18 perturbation of raft stability, 15e16 hypothesis, isolation, data interpretation, 10 detergent-based method, detergent-free method, 7e9 immunoblotting, 10 localization of GPCRs in, 6e7, 11e12 adherent cells, 14e15 cells in suspension, 13e14 confocal microscopy, 12e13 CTxB labeling, 11e12 D1R, 11f lipid rafts isolation, 7e10 membrane microdomain, 5f microdomains, 4e5 strategies to disrupt, 17t Live cell imaging, 29 Localization microscopy, 59 Locked nucleic acids (LNA), 156e157 LPA, see Lysophosphatidic acid (LPA) Luciferase (LUC), 237, 257e258 luciferase and DnaEN (lucN-DnaEN), 297 Luteinizing hormone receptor (LHR), 58, 266e269 Lysophosphatidic acid (LPA), 171 M M-CSF, see Macrophage colony stimulating factor (M-CSF) M1R, see Muscarinic M1 receptor (M1R) M2R, see Muscarinic M2 receptor (M2R) M3R, see Muscarinic M3 receptor (M3R) Macrophage colony stimulating factor (M-CSF), 67 Manually validated QuantiTect qPCR primer assays, 86te96t MAPK, see Mitogen-activated protein kinase (MAPK) MARTINI model, 435 MAS1 oncogene, 342e343 Mast cells (MCs), 175 Index MBS, see Mes-buffered solution (MBS) MCs, see Mast cells (MCs) MD, see Molecular dynamics (MD) Mechanistic models, 435e436 MeckeleGruber syndrome (MKS), 36e37 Melatonin (MT1), 266e269 Membrane lipid interactions, 443e445 Membrane protein threader (MP-T), 363 Mes, see 2-N-morpholino ethanesulfonic acid (Mes) Mes-buffered solution (MBS), 2-methyl-2-F-anandamide (Met-F-AEA), 175 Methyl-a-cyclodextrin (a-MCD), 7e8 Methyl-b-cyclodextrin (b-MCD), Methylation of the indol nitrogen of serotonin (MetI), 375e376 Microarray, 74 m-opioid receptor (OPRM), 369, 415 mOR, see m-opioid receptor (OPRM) Microscopic system, 112e113 Microtubules, 166e167 Migration, 174 Mitogen-activated protein kinase (MAPK), 321 MKS, see MeckeleGruber syndrome (MKS) MM, see Molecular mechanics (MM) Model refinement, 363e364 Model validation, 364 Modeling biased agonism, 372e373 Modulation binding assays, see Indirect binding assays MOE, see Molecular Operating Environment (MOE) Molecular “feedback” mechanism, Molecular docking, 364e367 Molecular dynamics (MD), 382e383, 406 simulations, 361, 378e379 disadvantage, 382e383 milestones of GPCRs, 367e370 Molecular mechanics (MM), 433 Molecular Operating Environment (MOE), 365e366 Molecular orbitals (MOs), 433 MOR, see Mu opioid receptor (MOR) 2-N-morpholino ethanesulfonic acid (Mes), Morpholino oligonucleotides (MOs), 150, 151t MOs, see Molecular orbitals (MOs); Morpholino oligonucleotides (MOs) Mouse PAR3 (mPAR3), 344 MP-specific substitution matrices (JSUBST matrices), 363 MP-T, see Membrane protein threader (MP-T) mPAR3, see Mouse PAR3 (mPAR3) MS-222, see Tricaine MT1, see Melatonin (MT1) Mu opioid receptor (MOR), 26e27 Multiscale simulations, 432e433 continuum electrostatic models, 436 mechanistic models, 435e436 phenomenological models, 436e437 QM models, 433 time-and length scales, 432f Muscarinic M1 receptor (M1R), 258e259 Muscarinic M2 receptor (M2R), 415 Muscarinic M3 receptor (M3R), 415 Mutual information, 379e380 N N-body information theory (NbIT), 379e380 n-Dodecyl b-D-maltoside (DDM), 122, 220 N/OFQ opioid receptor (OPRX), 415 NAMD, 368 NbIT, see N-body information theory (NbIT) NE, see Neutrophil elastase (NE) Nearest neighborhood approach, 66 Neomycin, see G418 Neurokinin type (NK1R), 266e269 Neutrophil elastase (NE), 348 NK1R, see Neurokinin type (NK1R) NLS, see Nuclear localization sequence (NLS) NMA, see Normal mode analysis (NMA) NMs, see Normal modes (NMs) Nonspecific binding (NSB), 193e194 Nonsynonymous single nucleotide polymorphisms (nsSNPs), 440 Nonvisual arrestins, 234e236 Normal mode analysis (NMA), 378, 383 Normal modes (NMs), 383 Nostoc punctiforme DnaE intein (NpuDnaE), 296 NSB, see Nonspecific binding (NSB) nsSNPs, see Nonsynonymous single nucleotide polymorphisms (nsSNPs) Nuclear ERK activation measurement using BRET-based ERK-NLS sensor in endogenous cell model experimental workflow, 332 results, 332e334 theory, 330e332 using BRET-based ERK-NLS sensor in heterologous cell models experimental workflow, 328 475 476 Index Nuclear ERK activation measurement (Continued) results, 328e330 theory, 328 Nuclear localization sequence (NLS), 321e322 Number of particles, volume, and pressure (NVT), 373e374 O OBPs, see Odorant-binding proteins (OBPs) ODE, see Ordinary differential equation- (ODE) Odorant-binding proteins (OBPs), 131 Odorant-receptors (ORs), 41, 128 Odorant(s), 131 adaptation mechanisms, 133e135 responses, 130e131, 134 Olfactory epithelium (OE), 41e42 Olfactory receptor signaling, 128 dimerization, 130 odorant adaptation mechanisms, 133e135 olfactory transduction, 131e133 perception, 130e131 perspectives, 135 specificity, 129e130 Olfactory receptors (OR), 371 Olfactory sensory neurons (OSNs), 40f, 41e42, 130e131, 133 in cilia GPCR, 41e45 Olfactory transduction, 131e133 Oligomerization, 56e57, 62e63, 68 OPRD, see d-opioid receptor (OPRD) OPRK, see k-opioid receptor (OPRK) OPRM, see m-opioid receptor (OPRM) OPRX, see N/OFQ opioid receptor (OPRX) OR, see Olfactory receptors (OR) Ordinary differential equation- (ODE), 436e437 Orexin (OX1), 266e269 ORs, see Odorant-receptors (ORs) Orthosteric binding site, 404 OSNs, see Olfactory sensory neurons (OSNs) OX1, see Orexin (OX1) P P-Rex1, 174 PALM, see Photoactivated localization microscopy (PALM) PAR, see Protease-activated receptors (PAR) Parathyroid hormone (PTH), 110e112, 111f Parathyroid hormone receptor (PTHR), 110e112, 111f, 259e261 PARS, see Prediction of Allosteric and Regulatory Sites (PARS) Partial differential equation- (PDE), 436e437 Patched (PTCH), 45 PB methods, see PoissoneBoltzmann methods (PB methods) PBS, see Phosphate buffer saline (PBS) PCA, see Principal component analysis (PCA) PCH analysis, see Photon countinghistogram analysis (PCH analysis) PCR, see Polymerase chain reaction (PCR) PCs, see Principal components (PCs) PDB database, see Protein Data Bank database (PDB database) PDE, see Partial differential equation- (PDE); Phosphodiesterases (PDE) PEI, see Polyethylenimine (PEI) Peptide desalting, 123e124 Peptidylprolyl isomerase A (PPIA), 74 expression of, 84b qPCR preparation master mix for quantification in triplicates of expression of, 83t QPCR quantification of GPCR expression relative adipose tissue housekeeping gene, 75e76, 97e98 relative housekeeping gene, 81e100 Perception, 130e131 PH domain, see Pleckstrin homology domain (PH domain) PH-domain-binding motifs in cancer, 350f PARs signaling endowing, 346e349, 350f Phalloidin-based protocol for actin filaments staining, 179e181 Pharmacology, 415e417 PHAT matrices, see Protein specific substitution matrices (PHAT matrices) Phenomenological models, 436e437 Phenylthiocarbamide (PTC), 414 Phosphate buffer saline (PBS), 220e221, 241 Phosphodiesterases (PDE), 110e112 Phospholipase C (PLC), 169 Photoactivated dye-localization microscopy, 61 counting single molecules custom quantification of GPCR monomers, dimers, and oligomers, 66e67 resolving single-molecule data, 65e66 seeing single molecules choice of probe and labeling, 61e62 imaging considerations and conditions, 63e65 optimization steps, 62e63 Photoactivated localization microscopy (PALM), 12e13, 57 Index Photon countinghistogram analysis (PCH analysis), 58 Photoreceptors, 45 Piecemeal secretion, 175e176 PKA, see Protein kinase A (PKA) PKD, see Polycystic kidney disease (PKD) Placental syndromes, 351 Plasma membrane, Plasmid vectors construction for assay system GPCReRab5 interaction, 302e303 GPCReb-arrestin interaction, 302 Plasmids, receptor expressing, 323 PLC, see Phospholipase C (PLC) Pleckstrin homology domain (PH domain), 346 PLUMED software plugin, 374e375 PNS, see Postnuclear supernatant (PNS) PoissoneBoltzmann methods (PB methods), 436 Polycystic kidney disease (PKD), 36e37 Polyethylenimine (PEI), 322 Polymerase chain reaction (PCR), 302 Pooled adipose tissue cDNA, 82e85 Postnuclear supernatant (PNS), 121 PP5, see Protein phosphatase type (PP5) PPIA, see Peptidylprolyl isomerase A (PPIA) PR, see Progesterone receptor (PR) PRALINETM, 363 Prediction of Allosteric and Regulatory Sites (PARS), 378 Preterm delivery (PTD), 351 Primary cilium, 36e37, 36f Principal component analysis (PCA), 378e379 Principal components (PCs), 378e379 PROCHECK, 364 ProDy, 378e379 Progesterone receptor (PR), 342 Protease-activated receptors (PAR), 176, 343e344, see also G protein-coupled receptors (GPCRs) ERK1/2 signaling, 345 and human placenta, 350e352 PAR1, 415 PAR1/PAR3 heterodimerization, 344 PH-domain-binding motifs, 346e349 in cancer, 350f Protein dynamics, 370 samples, 221e222 splicing, 295e296, 312e313 trans-splicing, 295e296 Protein Data Bank database (PDB database), 362e363 Protein kinase A (PKA), 168e169 Protein phosphatase type (PP5), 169 Protein specific substitution matrices (PHAT matrices), 363 Protein-ligand docking, 366 interaction identification tool, 364e367 Proteinases, 343 Proteineprotein docking, 381e382 interaction, 346e349 Proximity-based assays, 237 PTC, see Phenylthiocarbamide (PTC) PTCH, see Patched (PTCH) PTD, see Preterm delivery (PTD) PTH, see Parathyroid hormone (PTH) PTHR, see Parathyroid hormone receptor (PTHR) Q QM models, see Quantum mechanical models (QM models) qPCR, see quantitative real-time PCR (qPCR) qPCR analysis, 156e160, 158t assay design, 157 data analysis, 159e160 pipetting scheme for, 158f qPCR setup, 157e158 setting up cycler, 159 QSAR, see Quantitative structureeactivity relationship (QSAR) Quantitative analysis of G-protein-coupled receptor internalization, 295, 306e311 Firefly luciferase assay, 308e311 inteins, 295e296 materials and methods, 296e311 comparison of wild-type GPCR, 301f fusion expression constructs generation, 300e303 HEK 293 cells generation, 303e306 NpuDnaE, 296 reconstructed plasmid, 298f Renilla luciferase assay, 306e308 quantitative real-time PCR (qPCR), 74 quantification of GPCR expression materials, 75e76 methods, 81e100 Quantitative structureeactivity relationship (QSAR), 361, 370 Quantum effects, 437e438 Quantum mechanical models (QM models), 432e437 477 478 Index R Rab5, see Ras-associated protein (Rab5) Radioligand, 204e207 Rap1, 172e173 Rap2, 172e173 RAS, see Rennin-angiotensin system (RAS) Ras-associated protein (Rab5), 296 Rational design, 403e404 of biased molecules, 375e376 Rationale, 320e322 and objectives, 321e322 Reagents, 219e220 Receptor conformation analysis assays, 237 internalization, 269e270 oligomerization, 270e277 preparation, 202e204 receptor-bound radioligand separation, 208e210 receptor-expressing plasmids, 220 receptor-ligand binding assays, 192 Recycling, 26e27, 29e30 Redistribution-based assays, 236e237 Regulators of G protein signaling (RGS), 167e168 Relative luminescence units (RLU), 245 Renilla luciferase (Rluc), 237, 296, 299f assay, 306e308 Rennin-angiotensin system (RAS), 342e343 Repeated pregnancy loss (RPL), 351 Residue 6.54, 414 Resonance energy transfer (RET), 237, 321e322 Resonance energy transfer-based approaches (RET-based approaches), 256e258 b-arrestin recruitment, 266e269 GPCReG protein interaction, 261e265, 263f ligand binding, 258e259 ligand-induced conformational changes, 259e261, 261f receptor internalization, 269e270 receptor oligomerization, 270e277 second messenger production, 265e266 RET, see Resonance energy transfer (RET) RET-based approaches, see Resonance energy transfer-based approaches (RET-based approaches) Reverse transcription of RNA into cDNA, 156 RGS, see Regulators of G protein signaling (RGS) Rho-kinase (ROCK), 174 RhoA-mediated pathways, 171 Rhodopsin as ciliary GPCR, 38e39 oligomerization, 445e446 RLU, see Relative luminescence units (RLU) Rluc, see Renilla luciferase (Rluc) RlucC, see C-terminal domain of Renilla luciferase (RlucC) RNA isolation, 155e156 RNA-sequencing, 74 ROCK, see Rho-kinase (ROCK) Ropinirole hydrochloride, 249e250 RPL, see Repeated pregnancy loss (RPL) S S1P, see Sphingosine-1-phosphate (S1P) S1P1R/Gi signaling, 175 SA, see Surface area (SA) SANDOCK, 365e366 “Sandwich” labeling approach, 62 SAR, see Structureeactivity relationship (SAR) Saturation binding assays, 195e196 Scatchard transformations, 211 Schwann cells (SCs), 176e177 Scintillation proximity assays (SPA), 209e210 Screening assays, 236e237 SCs, see Schwann cells (SCs) SDS-PAGE, see SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-polyacrylamide gel electrophoresis (SDS-PAGE), 221e222 Second messenger production, 265e266 Secretion, 175 Semiemperical methods, 433 Sensor design, 326 Sensor validation, 326e327 SEP, see Super ecliptic phluorin (SEP) Sequence alignments, 404e410, 405t Sequence-based methods, 381 Serotonin receptor 1B (5HT1B), 415 Serotonin receptor 2B (5HT2B), 415 Serum response factor (SRF), 177 Server for Predicting Allosteric Communication and Effects of Regulation (SPACER), 378 Seven-transmembrane proteins (7-TM proteins), 167, 402 Shh, see Sonic Hedgehog (Shh) Signal transduction, Signaling assays EPAC assay for cAMP levels, 222e223 ERK1/2 MAP kinase activation, 222 complexes, 219, 442 events, 320e321 Signaling biosensors, 320e321 BRET measurements, 325e326 Index experimental strategies nuclear ERK activation measurement, 328e334 sensor design, 326 sensor validation, 326e327 materials and instrumentation biosensor and receptor plasmid construction, 323 instrumentation, 323 reagents, 322e323 rationale, 320e322 and objectives, 321e322 tissue culture and transfection, 323e325 Single molecules counting custom quantification of GPCR monomers, dimers, and oligomers, 66e67 resolving single-molecule data, 65e66 seeing choice of probe and labeling, 61e62 imaging considerations and conditions, 63e65 optimization steps, 62e63 Slow odorant adaptation, 133e134 Smoothened receptor (SMO), 45, 415 Smoothened-mediated HH signaling analysis in zebrafish, 150e160 manipulating hedgehog signaling in zebrafish, 150e153 antisense MOs, 150 delivery of antisense MOs, 151e153 injection setup for zebrafish embryos, 152f smoothened inhibition by small chemical compounds, 153 transcript analysis of hedgehog signaling in zebrafish, 155e160 qPCR analysis, 156e160 reverse transcription of RNA into cDNA, 156 RNA isolation, 155e156 whole-mount analysis of hedgehog signaling in zebrafish, 153e155 Somatostatin receptor (SSTR2), 272e274 Sonic Hedgehog (Shh), 148 SPA, see Scintillation proximity assays (SPA) SPACER, see Server for Predicting Allosteric Communication and Effects of Regulation (SPACER) Spatial intensity distribution analysis (SpIDA), 58 Specific binding, 193e194 Specificity, 129e130 Sphingosine-1-phosphate (S1P), 171, 174e175 SpIDA, see Spatial intensity distribution analysis (SpIDA) Splice-blocking MOs, 150 SRF, see Serum response factor (SRF) Ssp, see Synechocystis sp strain PCC6803 (Ssp) SSTR2, see Somatostatin receptor (SSTR2) State-of-the-art experimental approaches, 431 State-of-the-art simulation methods, 432e433 Stimulated emission depletion (STED), 59 Stochastic models, 436e437 Stochastic optical reconstruction microscopy (STORM), 12e13, 59 Structural information, 373 Structure-based methods, 381 electrostatics studies, 383e384 molecular dynamics, 382e383 NMA, 383 proteineprotein docking, 381e382 Structure-based pharmacophore modeling, 414 Structureeactivity relationship (SAR), 370e371 Sucrose gradient ultracentrifugation, Super ecliptic phluorin (SEP), 27 Super-resolution imaging of single molecules, 59e61 Surface area (SA), 436 Survival, 176 Synechocystis sp strain PCC6803 (Ssp), 295e296 T T lymphocytes, 174 TAARs, see Trace amino acid receptors (TAARs) Tango assay for ligand-induced GPCRebarrestin2 interaction, 234e236 background and rationale, 234e236 b-arrestin recruitment assays, 236e239 data analysis, 245e247 GPCR signaling, 235f molecular basis of b-arrestin Tango assay, 241f principle of Tango assay, 239e240 reagents and methods, 240e245 cell line revival and maintenance, 242 cell trypsinization, 242e243 drug plate preparation and drug addition, 243e245 transient transfection in HTLA cell line, 243 results and data interpretation, 247e250 Taq polymerase, 157 TAS2Rs, 403 TAS2R-specific conserved residues, 409 TCS, see TEV protease cleavage site (TCS) Ternary complex model, 360e361 Tetracycline-controlled transactivator (tTA), 239e240 479 480 Index Tetracycline-inducible HEK 293 Flp-In T-Rex cell line, 219 TEV, see Tobacco etch virus (TEV) TEV protease cleavage site (TCS), 239e240 TFA, see Trifluoroacetic acid (TFA) TGF-b signaling pathways, 177 Thrombin, 176 receptor, 343 Time-resolved fluorescence resonance energy transfer (TR-FRET), 257e258 TIRF FRET, 113 microscopic system, 113 TIRF microscopy, see Total internal reflection fluorescence microscopy (TIRF microscopy) TIRF-M, see Total internal reflection fluorescent microscopy (TIRF-M) Tissue culture, 220 7-TM proteins, see Seven-transmembrane proteins (7-TM proteins) TM receptors, see Transmembrane receptors (TM receptors) Tobacco etch virus (TEV), 239e240 Toggle switch, 407 Total correlation, 379e380 Total internal reflection fluorescence microscopy (TIRF microscopy), 26e27, 112 image analysis, 30e31 imaging cell culture, 28e29 live cell imaging, 29 important considerations before imaging, 27e28 materials cell culture, 28 TIRF microscopy equipment and settings, 28 notes, 30 Total internal reflection fluorescent microscopy (TIRF-M), 57 TR-FRET, see Time-resolved fluorescence resonance energy transfer (TR-FRET) Trace amino acid receptors (TAARs), 41 Transcript analysis of hedgehog signaling in zebrafish, 155e160 qPCR analysis, 156e160 reverse transcription of RNA into cDNA, 156 RNA isolation, 155e156 Transition zone, 36e37 Translation-blocking MOs, 150 Transmembrane receptors (TM receptors), 361 Transmission switch, 407 Tricaine, 155 Trifluoroacetic acid (TFA), 124 “Triple-negative” breast cancers, 342 tTA, see Tetracycline-controlled transactivator (tTA) U Universal Probe System, 156e157 Universal probes (UP), 156e157 V V2R, See V2 vasopressin receptor (V2R) van der Waals and coulombic interactions, 435 Vasodilator-stimulated phosphoprotein (VASP), 173 Vasopressin V2 (V2R), 259e261, 266e269, 323 Vav3 proto-oncogene, 348 VERIFY3D, 364 Vertebrate GPCRs, 167 Virtual screening (VS), 361, 371e372 Visual arrestins, 234e236 W Western blot method, 10 Western blotting, 220e222 Whole-mount analysis of hedgehog signaling in zebrafish, 153e155 Wide-field FRET, 112e113 microscopic system, 112e113 Wild-type LHR (WT LHR), 62 Y Yellow fluorescent protein (YFP), 237 You-mutants, 150 Z Zebrafish, 148e149 ... experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety... order by intertwining into the hydrophobic gaps between the phospholipid acyl chains Certain structural proteins abound in lipid rafts to serve as scaffold or anchor for other proteins, including... detailed in Boesze-Battaglia, 2006) Other pore-forming toxins, besides CTxB, used to visualize lipid rafts include equinatoxin II which binds dispersed sphingomyelin, lysenin which binds clustered