Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist‐Induced Arrestin Recruitment to Modified and Unmodified G Protein‐Coupled Receptors

Prashant Donthamsetti1, Jose Rafael Quejada1, Jonathan A. Javitch1, Vsevolod V. Gurevich2, Nevin A. Lambert3

1 Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, 2 Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, 3 Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.14
DOI:  10.1002/0471141755.ph0214s70
Online Posting Date:  September, 2015
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G protein‐coupled receptors (GPCRs) represent ∼25% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which are involved in differential signaling pathways. Because functionally selective or biased ligands activate one of these two pathways, they may be superior medications for certain diseases states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for two bioluminescence resonance energy transfer (BRET)‐based assays used to monitor arrestin recruitment to GPCRs. One assay requires modification of GPCRs by fusion to a BRET donor or acceptor moiety, whereas the other can detect arrestin recruitment to unmodified GPCRs. © 2015 by John Wiley & Sons, Inc.

Keywords: G protein‐coupled receptors (GPCRs); arrestin; bioluminescence resonance energy transfer (BRET)

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Table of Contents

  • Introduction
  • Basic Protocol 1: Receptor‐Arrestin BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Receptors
  • Alternate Protocol 1: Arrestin Translocation BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Plasma Membrane
  • Support Protocol 1: Optimization of Polyethylenimine (PEI) Concentration for Transfection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Receptor‐Arrestin BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Receptors

  • HEK293T cells (ATCC, cat. no. CRL‐3216), fully confluent 10‐cm plate
  • HEK293T culture medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; Cellgro, cat. no. 21‐031‐CV)
  • Trypsin (Cellgro, cat. no. 25‐052‐Cl)
  • Mammalian expression plasmids (plasmids that are not commercially available can be obtained from the authors for non‐profit use):
    • Plasmid encoding donor‐fused GPCR, e.g., pcDNA3.1‐D2R‐linker‐Rluc8
    • Plasmid encoding acceptor‐fused arrestin, e.g., pIRES‐puro‐Venus‐linker‐arrestin‐3
    • Plasmid encoding GRK, e.g., pcDNA3.1‐GRK2
    • Empty vector plasmid, e.g., pcDNA3.1 (Life Technologies)
  • DMEM (Gibco, cat. no. 11965‐092)
  • 1 μg/μl polyethyleimine (PEI; see recipe)
  • Agonist stock: e.g., 22 mM dopamine in dH 2O (dopamine hydrochloride available from Sigma‐Aldrich, cat. no. H8502)
  • Antagonist stock: e.g., 44 mM sulpiride in DMSO (S‐(−)‐sulpiride available from Sigma‐Aldrich, cat. no. S7771)
  • DPBS with 40 mg/L sodium bisulfite, pH 7.4, used to reduce dopamine oxidation (required only when using dopamine)
  • 5 mM glucose in DPBS, pH 7.4
  • 5 mM coelenterazine H in absolute ethanol (see recipe)
  • 10‐cm tissue‐culture plates (BD Falcon, cat. no. 353003)
  • Compound plate, 96‐well V‐bottom plates (Greiner Bio‐One, cat. no. 651101)
  • BRET assay plate, white 96‐well flat bottom plates (Greiner Bio‐One, cat. no. 655075) or Black/White 96‐well Isoplate (PerkinElmer, cat. no. 6005030)
  • 12‐Channel multichannel pipettor, 50‐300 μl (Labsystems Finnpipette, cat. no. Z368989) or 5‐50 μl (Labsystems Finnpipette, cat. no. Z678031)
  • Pipette basin (USA Scientific, cat. no. 2320‐2620)
  • Repeater Plus Pipettor (Eppendorf, cat. no. 022260201)
  • Plate reader for luminescence, fluorescence, and BRET detection (e.g., BMG Labtech, Pherastar FS, or Tecan, Infinite F500
  • Software for data analysis (e.g., Microsoft Excel and GraphPad Prism)
NOTE: This protocol can be employed for either a single dopamine agonist curve or a sulpiride antagonist curve at a single dose of dopamine. The procedure can be scaled up, depending on the number of compounds for screening.NOTE: All mammalian tissue culture must be conducted using aseptic techniques in a laminar flow hood. Cells should be maintained in an incubator at 37°C at 5% CO 2.

Alternate Protocol 1: Arrestin Translocation BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Plasma Membrane

  Additional Materials (also see protocol 1Basic Protocol)
  • Mammalian expression plasmids:
    • Plasmid encoding for GPCR (e.g., pcDNA3.1‐D2R)
    • Plasmid encoding for Rluc8‐Arrestin‐3‐Sp1
    • Plasmid encoding for mem‐linker‐citrine‐SH3

Support Protocol 1: Optimization of Polyethylenimine (PEI) Concentration for Transfection

  • See protocol 1Basic Protocol
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Literature Cited

Literature Cited
  Ahn, S., Shenoy, S.K., Wei, H., and Lefkowitz, R.J. 2004. Differential kinetic and spatial patterns of beta‐arrestin and G protein‐mediated ERK activation by the angiotensin II receptor. J. Biol. Chem. 279:35518‐35525.
  Atwood, B.K., Lopez, J., Wager‐Miller, J., Mackie, K., and Straiker, A. 2011. Expression of G protein‐coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis. BMC Genomics 12:14.
  Celver, J., Vishnivetskiy, S.A., Chavkin, C., and Gurevich, V.V. 2002. Conservation of the phosphate‐sensitive elements in the arrestin family of proteins. J. Biol. Chem. 277:9043‐9048.
  Charest, P.G., Terrillon, S., and Bouvier, M. 2005. Monitoring agonist‐promoted conformational changes of beta‐arrestin in living cells by intramolecular BRET. EMBO Rep. 6:334‐340.
  Clayton, C.C., Donthamsetti, P., Lambert, N.A., Javitch, J.A., and Neve, K.A. 2014. Mutation of three residues in the third intracellular loop of the dopamine D2 receptor creates an internalization‐defective receptor. J. Biol. Chem. 289:33663‐33675. doi: 10.1074/jbc.M114.605378.
  Coffa, S., Breitman, M., Spiller, B.W., and Gurevich, V.V. 2011. A single mutation in arrestin‐2 prevents ERK1/2 activation by reducing c‐Raf1 binding. Biochemistry 50:6951‐6958.
  Eishingdrelo, H. and Kongsamut, S. 2013. Minireview: Targeting GPCR Activated ERK Pathways for Drug Discovery. Curr. Chem. Genomics Transl. Med. 7:9‐15.
  Gimenez, L.E., Babilon, S., Wanka, L., Beck‐Sickinger, A.G., and Gurevich, V.V. 2014. Mutations in arrestin‐3 differentially affect binding to neuropeptide Y receptor subtypes. Cell Signal. 26:1523‐1531.
  Gimenez, L.E., Kook, S., Vishnivetskiy, S.A., Ahmed, M.R., Gurevich, E.V., and Gurevich, V.V. 2012a. Role of receptor‐attached phosphates in binding of visual and non‐visual arrestins to G protein‐coupled receptors. J. Biol. Chem. 287:9028‐9040.
  Gimenez, L.E., Vishnivetskiy, S.A., Baameur, F., and Gurevich, V.V. 2012b. Manipulation of very few receptor discriminator residues greatly enhances receptor specificity of non‐visual arrestins. J. Biol. Chem. 287:29495‐29505.
  Grunberg, R., Burnier, J.V., Ferrar, T., Beltran‐Sastre, V., Stricher, F., van der Sloot, A.M., Garcia‐Olivas, R., Mallabiabarrena, A., Sanjuan, X., Zimmermann, T., and Serrano, L. 2013. Engineering of weak helper interactions for high‐efficiency FRET probes. Nat. Methods 10:1021‐1027.
  Gurevich, V.V. 1998. The selectivity of visual arrestin for light‐activated phosphorhodopsin is controlled by multiple nonredundant mechanisms. J. Biol. Chem. 273:15501‐15506.
  Gurevich, V.V. and Gurevich, E.V. 2006. The structural basis of arrestin‐mediated regulation of G protein‐coupled receptors. Pharm. Ther. 110:465‐502.
  Hamdan, F.F., Audet, M., Garneau, P., Pelletier, J., and Bouvier, M. 2005. High‐throughput screening of G protein‐coupled receptor antagonists using a bioluminescence resonance energy transfer 1‐based beta‐arrestin2 recruitment assay. J. Biomol. Screen. 10:463‐475.
  Hamdan, F.F., Percherancier, Y., Breton, B., and Bouvier, M. 2006. Monitoring protein‐protein interactions in living cells by bioluminescence resonance energy transfer (BRET). Curr. Protoc. Neurosci. 34:5.23.1‐5.23.20.
  Hirsch, J.A., Schubert, C., Gurevich, V.V., and Sigler, P.B. 1999. The 2.8 Å crystal structure of visual arrestin: A model for arrestin's regulation. Cell 97:257‐269.
  Kim, Y.M. and Benovic, J.L. 2002. Differential roles of arrestin‐2 interaction with clathrin and adaptor protein 2 in G protein‐coupled receptor trafficking. J. Biol. Chem. 277:30760‐30768.
  Klewe, I.V., Nielsen, S.M., Tarpo, L., Urizar, E., Dipace, C., Javitch, J.A., Gether, U., Egebjerg, J., and Christensen, K.V. 2008. Recruitment of beta‐arrestin2 to the dopamine D2 receptor: Insights into anti‐psychotic and anti‐parkinsonian drug receptor signaling. Neuropharmacology 54:1215‐1222.
  Kocan, M. and Pfleger, K.D. 2011. Study of GPCR‐protein interactions by BRET. Methods Mol. Biol. 746:357‐371.
  Kovoor, A., Celver, J., Abdryashitov, R.I., Chavkin, C., and Gurevich, V.V. 1999. Targeted construction of phosphorylation‐independent b‐arrestin mutants with constitutive activity in cells. J. Biol. Chem. 274:6831‐6834.
  Lefkowitz, R.J. 2013. Arrestins come of age: A personal historical perspective. Prog. Mol. Biol. Transl. Sci. 118:3‐18.
  Loening, A.M., Fenn, T.D., Wu, A.M., and Gambhir, S.S. 2006. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng. Des. Sel. 19:391‐400.
  Marullo, S. and Bouvier, M. 2007. Resonance energy transfer approaches in molecular pharmacology and beyond. Trends Pharmacol. Sci. 28:362‐365.
  Morin, J.G. and Hastings, J.W. 1971. Energy transfer in a bioluminescent system. J. Cell. Physiol. 77:313‐318.
  Morise, H., Shimomura, O., Johnson, F.H., and Winant, J. 1974. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 13:2656‐2662.
  Nobles, K.N., Guan, Z., Xiao, K., Oas, T.G., and Lefkowitz, R.J. 2007. The active conformation of beta‐arrestin1: Direct evidence for the phosphate sensor in the N‐domain and conformational differences in the active states of beta‐arrestins1 and ‐2. J. Biol. Chem. 282:21370‐21381.
  Overington, J.P., Al‐Lazikani, B., and Hopkins, A.L. 2006. How many drug targets are there? Nat. Rev. Drug. Discov. 5:993‐996.
  Pfleger, K.D., Seeber, R.M., and Eidne, K.A. 2006. Bioluminescence resonance energy transfer (BRET) for the real‐time detection of protein‐protein interactions. Nat. Protoc. 1:337‐345.
  Salahpour, A., Espinoza, S., Masri, B., Lam, V., Barak, L.S., and Gainetdinov, R.R. 2012. BRET biosensors to study GPCR biology, pharmacology, and signal transduction. Front. Endocrinol. 3:105.
  Shukla, A.K., Manglik, A., Kruse, A.C., Xiao, K., Reis, R.I., Tseng, W.C., Staus, D.P., Hilger, D., Uysal, S., Huang, L.Y., Paduch, M., Tripathi‐Shukla, P., Koide, A., Koide, S., Weis, W.I., Kossiakoff, A.A., Kobilka, B.K., and Lefkowitz, R.J. 2013. Structure of active beta‐arrestin‐1 bound to a G‐protein‐coupled receptor phosphopeptide. Nature 497:137‐141.
  van der Lee, M.M., Blomenrohr, M., van der Doelen, A.A., Wat, J.W., Smits, N., Hanson, B.J., van Koppen, C.J., and Zaman, G.J. 2009. Pharmacological characterization of receptor redistribution and beta‐arrestin recruitment assays for the cannabinoid receptor 1. J. Biomol. Screen. 14:811‐823.
  Violin, J.D., Crombie, A.L., Soergel, D.G., and Lark, M.W. 2014. Biased ligands at G‐protein‐coupled receptors: Promise and progress. Trends Pharmacol. Sci. 35:308‐316.
  Vishnivetskiy, S.A., Gimenez, L.E., Francis, D.J., Hanson, S.M., Hubbell, W.L., Klug, C.S., and Gurevich, V.V. 2011. Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins. J. Biol. Chem. 286:24288‐24299.
  Vishnivetskiy, S.A., Paz, C.L., Schubert, C., Hirsch, J.A., Sigler, P.B., and Gurevich, V.V. 1999. How does arrestin respond to the phosphorylated state of rhodopsin? J. Biol. Chem. 274:11451‐11454.
  Wess, J., Nakajima, K., and Jain, S. 2013. Novel designer receptors to probe GPCR signaling and physiology. Trends Pharmacol. Sci. 34:385‐392.
  Zhang, R. and Xie, X. 2012. Tools for GPCR drug discovery. Acta Pharmacol. Sin. 33:372‐384.
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