Skip to main content
SpringerLink
Log in

Measurements of β-Arrestin Recruitment to Activated Seven Transmembrane Receptors Using Enzyme Complementation

  • Protocol
  • First Online:
Receptor Binding Techniques

Part of the book series: Methods in Molecular Biology ((MIMB,volume 897))

Abstract

The recruitment of arrestins to activated 7TMRs results in the activation of alternative signaling pathways, quenching of G-protein activation, and coupling to clathrin-mediated endocytosis. The nearly ubiquitous involvement of arrestin in 7TMR signaling has spurred the development of several methods for monitoring this interaction in mammalian cells. Nonetheless, few maintain the reproducibility and precision necessary for drug discovery applications. Enzyme fragment complementation technology (EFC) is an emerging protein–protein interaction technology based on the forced complementation of a split enzyme that has proven to be highly effective in monitoring the formation of GPCR–arrestin complexes. In these systems, the target proteins are fused to two fragments of an enzyme that show little or no spontaneous complementation. Interaction of the two proteins forces the complementation of the enzyme, resulting in an enzymatic measure of the protein interaction. This chapter discusses the utility and methods involved in using the PathHunter β-galactosidase complementation system to monitor arrestin recruitment and the advantages of exploiting this pathway in the characterization of 7TMR function.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fredriksson R, Lagerström MC, Lundin LG, Schiöth HB (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 63:1256–1272

    Article  PubMed  CAS  Google Scholar 

  2. Kenakin T (2011) Functional selectivity and biased receptor signaling. J Pharmacol Exp Ther 336:296–302

    Article  PubMed  CAS  Google Scholar 

  3. Williams C, Hill SJ (2009) GPCR signaling: understanding the pathway to successful drug discovery. Methods Mol Biol 552:39–50

    Article  PubMed  CAS  Google Scholar 

  4. Whalen EJ, Rajagopal S, Lefkowitz RJ (2011) Therapeutic potential of beta-arrestin- and G protein-biased agonists. Trends Mol Med 17:126–139

    Article  PubMed  CAS  Google Scholar 

  5. Rajagopal S, Rajagopal K, Lefkowitz RJ (2010) Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov 9:373–386

    Article  PubMed  CAS  Google Scholar 

  6. DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK (2007) Beta-arrestins and cell signaling. Annu Rev Physiol 69:483–510

    Article  PubMed  CAS  Google Scholar 

  7. Gurevich VV, Gurevich EV, Cleghorn WM (2008) Arrestins as multi-functional signaling adaptors. Handb Exp Pharmacol 186:15–37

    Article  PubMed  CAS  Google Scholar 

  8. Kovacs JJ, Hara MR, Davenport CL, Kim J, Lefkowitz RJ (2009) Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways. Dev Cell 17:443–458

    Article  PubMed  CAS  Google Scholar 

  9. Luttrell LM, Gesty-Palmer D (2010) Beyond desensitization: physiological relevance of arrestin-dependent signaling. Pharmacol Rev 62:305–330

    Article  PubMed  CAS  Google Scholar 

  10. Walters RW, Shukla AK, Kovacs JJ, Violin JD, DeWire SM, Lam CM, Chen JR, Muehlbauer MJ, Whalen EJ, Lefkowitz RJ (2009) beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice. J Clin Invest 119:1312–1321

    Article  PubMed  CAS  Google Scholar 

  11. Ferguson SS, Caron MG (2004) Green fluores­cent protein-tagged beta-arrestin translocation as a measure of G protein-coupled receptor activation. Methods Mol Biol 237:121–126

    PubMed  CAS  Google Scholar 

  12. Ferguson SS, Downey WE III, Colapietro AM, Barak LS, Ménard L, Caron MG (1996) Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science 271:363–366

    Article  PubMed  CAS  Google Scholar 

  13. Oakley RH, Hudson CC, Cruickshank RD, Meyers DM, Payne RE Jr, Rhem SM, Loomis CR (2002) The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors. Assay Drug Dev Technol 1:21–30

    Article  PubMed  CAS  Google Scholar 

  14. Garippa RJ, Hoffman AF, Gradl G, Kirsch A (2006) High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera. Methods Enzymol 414:99–120

    Article  PubMed  CAS  Google Scholar 

  15. Heding A (2004) Use of the BRET 7TM receptor/beta-arrestin assay in drug discovery and screening. Expert Rev Mol Diagn 4:403–411

    Article  PubMed  CAS  Google Scholar 

  16. van der Lee MM, Blomenröhr M, van der Doelen AA, Wat JW, Smits N, Hanson BJ, van Koppen CJ, Zaman GJ (2009) Pharmacological characterization of receptor redistribution and beta-arrestin recruitment assays for the cannabinoid receptor 1. J Biomol Screen 14:811–823

    Article  PubMed  Google Scholar 

  17. Verkaar F, van Rosmalen JW, Blomenröhr M, van Koppen CJ, Blankensteijn WM, Smits JF, Zaman GJ (2008) G protein-independent cell-based assays for drug discovery on seven-transmembrane receptors. Biotechnol Annu Rev 14:253–274

    Article  PubMed  CAS  Google Scholar 

  18. Cabantous S, Pédelacq JD, Mark BL, Naranjo C, Terwilliger TC, Waldo GS (2005) Recent advances in GFP folding reporter and split-GFP solubility reporter technologies. Application to improving the folding and solubility of recalcitrant proteins from Mycobacterium tuberculosis. J Struct Funct Genomics 6:113–119

    Article  PubMed  CAS  Google Scholar 

  19. Wehrman T, Kleaveland B, Her JH, Balint RF, Blau HM (2002) Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments. Proc Natl Acad Sci U S A 99:3469–3474

    Article  PubMed  CAS  Google Scholar 

  20. Galarneau A, Primeau M, Trudeau LE, Michnick SW (2002) Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat Biotechnol 20:619–622

    Article  PubMed  CAS  Google Scholar 

  21. Wehrman T, He X, Raab B, Dukipatti A, Blau H, Garcia KC (2007) Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron 53:25–38

    Article  PubMed  CAS  Google Scholar 

  22. Wehrman TS, Casipit CL, Gewertz NM, Blau HM (2005) Enzymatic detection of protein translocation. Nat Methods 2:521–527

    Article  PubMed  CAS  Google Scholar 

  23. von Degenfeld G, Wehrman TS, Hammer MM, Blau HM (2007) A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals. FASEB J 21:3819–3826

    Article  Google Scholar 

  24. Paulmurugan R, Gambhir SS (2005) Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal Chem 77:1295–1302

    Article  PubMed  CAS  Google Scholar 

  25. Casanova E, Lemberger T, Fehsenfeld S, Mantamadiotis T, Schütz G (2003) Alpha complementation in the Cre recombinase enzyme. Genesis 37:25–29

    Article  PubMed  CAS  Google Scholar 

  26. Sun YC, Li Y, Zhang H, Yan HQ, Dowling DN, Wang YP (2006) Reconstitution of the enzyme AroA and its glyphosate tolerance by fragment complementation. FEBS Lett 580:1521–1527

    Article  PubMed  CAS  Google Scholar 

  27. Djannatian MS, Galinski S, Fischer TM, Rossner MJ (2011) Studying G protein-coupled receptor activation using split-tobacco etch virus assays. Anal Biochem 412: 141–152

    Article  PubMed  CAS  Google Scholar 

  28. van Der Lee MM, Bras M, van Koppen CJ, Zaman GJ (2008) beta-Arrestin recruitment assay for the identification of agonists of the sphingosine 1-phosphate receptor EDG1. J Biomol Screen 13:986–998

    Article  Google Scholar 

  29. McGuinness D, Malikzay A, Visconti R, Lin K, Bayne M, Monsma F, Lunn CA (2009) Characterizing cannabinoid CB2 receptor ligands using DiscoveRx PathHunter beta-arrestin assay. J Biomol Screen 14:49–58

    Article  PubMed  CAS  Google Scholar 

  30. Patel A, Murray J, McElwee-Whitmer S, Bai C, Kunapuli P, Johnson EN (2009) A combination of ultrahigh throughput PathHunter and cytokine secretion assays to identify glucocorticoid receptor agonists. Anal Biochem 385:286–292

    Article  PubMed  CAS  Google Scholar 

  31. Gao ZG, Jacobson KA (2008) Translocation of arrestin induced by human A(3) adenosine receptor ligands in an engineered cell line: comparison with G protein-dependent pathways. Pharmacol Res 57:303–311

    Article  PubMed  CAS  Google Scholar 

  32. Rosethorne EM, Charlton SJ (2011) Agonist-biased signaling at the histamine H4 receptor: JNJ7777120 recruits {beta}-arrestin without activating G proteins. Mol Pharmacol 79:749–757

    Article  PubMed  CAS  Google Scholar 

  33. Poulin B, Butcher A, McWilliams P, Bourgognon JM, Pawlak R, Kong KC, Bottrill A, Mistry S, Wess J, Rosethorne EM, Charlton SJ, Tobin AB (2010) The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner. Proc Natl Acad Sci U S A 107:9440–9445

    Article  PubMed  CAS  Google Scholar 

  34. McPherson J, Rivero G, Baptist M, Llorente J, Al-Sabah S, Krasel C, Dewey WL, Bailey CP, Rosethorne EM, Charlton SJ, Henderson G, Kelly E (2010) mu-Opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization. Mol Pharmacol 78:756–766

    Article  PubMed  CAS  Google Scholar 

  35. Kong KC, Butcher AJ, McWilliams P, Jones D, Wess J, Hamdan FF, Werry T, Rosethorne EM, Charlton SJ, Munson SE, Cragg HA, Smart AD, Tobin AB (2010) M3-muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1. Proc Natl Acad Sci U S A 107:21181–21186

    Article  PubMed  CAS  Google Scholar 

  36. Zhao X, Jones A, Olson KR, Peng K, Wehrman T, Park A, Mallari R, Nebalasca D, Young SW, Xiao SH (2008) A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors. J Biomol Screen 13:737–747

    Article  PubMed  CAS  Google Scholar 

  37. Yin H, Chu A, Li W, Wang B, Shelton F, Otero F, Nguyen DG, Caldwell JS, Chen YA (2009) Lipid G protein-coupled receptor ligand identification using beta-arrestin PathHunter assay. J Biol Chem 284:12328–12338

    Article  PubMed  CAS  Google Scholar 

  38. Wehrman TS, Raab WJ, Casipit CL, Doyonnas R, Pomerantz JH, Blau HM (2006) A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions. Proc Natl Acad Sci U S A 103:19063–19068

    Article  PubMed  CAS  Google Scholar 

  39. Rajagopal S, Ahn S, Rominger DH, Gowen-Macdonald W, Lam CM, Dewire SM, Violin JD, Lefkowitz RJ (2011) Quantifying ligand bias at seven-transmembrane receptors. Mol Pharmacol 80:367–377

    Article  PubMed  CAS  Google Scholar 

  40. van Lith LH, Oosteromm J, van Elsas A, Zaman GJ (2009) C5a-stimulated recruitment of beta-arrestin2 to the nonsignaling 7-transmembrane decoy receptor C5L2. J Biomol Screen 14:1067–1075

    Article  PubMed  Google Scholar 

  41. Zabel BA, Wang Y, Lewén S, Berahovich RD, Penfold ME, Zhang P, Powers J, Summers BC, Miao Z, Zhao B, Jalili A, Janowska-Wieczorek A, Jaen JC, Schall TJ (2009) Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated tumor cell transendothelial migration by CXCR7 ligands. J Immunol 183:3204–3211

    Article  PubMed  CAS  Google Scholar 

  42. Waterfield A, Kinloch R, Williams R, Young T, Nickolls S (2009) The DiscoveRx PathHunter β-arrestin assay; a measure of agonist affinity? pA2online 3:001

    Google Scholar 

  43. Charlton SJ, Vauquelin G (2010) Elusive equilibrium: the challenge of interpreting receptor pharmacology using calcium assays. Br J Pharmacol 161:1250–1265

    Article  PubMed  CAS  Google Scholar 

  44. Adham N, Ellerbrock B, Hartig P, Weinshank RL, Branchek T (1993) Receptor reserve masks partial agonist activity of drugs in a cloned rat 5-hydroxytryptamine1B receptor expression system. Mol Pharmacol 43:427–433

    PubMed  CAS  Google Scholar 

  45. Masri B, Salahpour A, Didriksen M, Ghisi V, Beaulieu JM, Gainetdinov RR, Caron MG (2008) Antagonism of dopamine D2 receptor/beta-arrestin 2 interaction is a common property of clinically effective antipsychotics. Proc Natl Acad Sci U S A 105:13656–13661

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. DiscoveRx Corporation, Fremont, CA, USA

    Daniel L. Bassoni, William J. Raab, Philip L. Achacoso, Chin Yee Loh & Tom S. Wehrman

Authors
  1. Daniel L. Bassoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William J. Raab
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Philip L. Achacoso
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chin Yee Loh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tom S. Wehrman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tom S. Wehrman .

Editor information

Editors and Affiliations

  1. Addenbrooke's Hospital, Box 110, Clinical Pharmacology Unit, University of Cambridge, Hills Road, Cambridge, CB2 2QQ, United Kingdom

    Anthony P. Davenport

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Bassoni, D.L., Raab, W.J., Achacoso, P.L., Loh, C.Y., Wehrman, T.S. (2012). Measurements of β-Arrestin Recruitment to Activated Seven Transmembrane Receptors Using Enzyme Complementation. In: Davenport, A. (eds) Receptor Binding Techniques. Methods in Molecular Biology, vol 897. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-909-9_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-909-9_9

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-908-2

  • Online ISBN: 978-1-61779-909-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation