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A High-Content, Live-Cell, and Real-Time Approach to the Quantitation of Ligand-Induced β-Arrestin2 and Class A/Class B GPCR Mobilization

Published online by Cambridge University Press:  28 January 2013

Anthony P. Leonard ,
Kathryn M. Appleton ,
Louis M. Luttrell  and
Yuri K. Peterson
Anthony P. Leonard
Affiliation:
Medical Universityof South Carolina, Pharmaceutical and Biomedical Sciences, Charleston, SC 29425, USA
Kathryn M. Appleton
Affiliation:
Medical Universityof South Carolina, Pharmaceutical and Biomedical Sciences, Charleston, SC 29425, USA
Louis M. Luttrell
Affiliation:
Medical Universityof South Carolina, Medicine, Charleston, SC 29425, USA
Yuri K. Peterson*
Affiliation:
Medical Universityof South Carolina, Pharmaceutical and Biomedical Sciences, Charleston, SC 29425, USA
*
*Corresponding author. E-mail: [email protected]
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Abstract

We report the development of a method to analyze receptor and β-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged β2-adrenergic receptor (β2-AR) or parathyroid hormone receptor type 1 (PTH1R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged β-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, β-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to β-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting β-arrestin interaction found with β2-AR agonist stimulation, in contrast to stronger mobilization and receptor colocalization with agonist stimulation of the PTH1R. Though especially appropriate for receptor kinetic studies, this method is generalizable to any dual fluorescence probe system in which quantification of object formation and movement is desired. These methodologies allow for quantitative, unbiased measurement of microscopy data and are further enhanced by providing real-time kinetics.

Keywords

high contentGPCRadrenergicparathyroidarrestincolocalizationkineticsimage processingmicroscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 150 - 170
Copyright
Copyright © Microscopy Society of America 2013

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Footnotes

†

These authors contributed equally to this work.

References

Atwood, B.K., Lopez, J., Wager-Miller, J., Mackie, K. & 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.Google Scholar
Barker, B.L. & Benovic, J.L. (2011). G protein-coupled receptor kinase 5 phosphorylation of hip regulates internalization of the chemokine receptor CXCR4. Biochemistry 50(32), 6933–6941.Google Scholar
Bockaert, J., Perroy, J., Becamel, C., Marin, P. & Fagni, L. (2010). GPCR interacting proteins (GIPs) in the nervous system: Roles in physiology and pathologies. Annu Rev Pharmacol Toxicol 50, 89–109.Google Scholar
Claing, A. & Laporte, S.A. (2005). Novel roles for arrestins in G protein-coupled receptor biology and drug discovery. Curr Opin Drug Discov Devel 8(5), 585–589.Google Scholar
Dale, L.B. & Ferguson, S.S. (2011). Simultaneous real-time imaging of signal oscillations using multiple fluorescence-based reporters. Methods Mol Biol 756, 273–281.CrossRefGoogle ScholarPubMed
Dorval, T., Ogier, A., Genovesio, A., Lim, H.K., Kwon do, Y., Lee, J.H., Worman, H.J., Dauer, W. & Grailhe, R. (2010). Contextual automated 3D analysis of subcellular organelles adapted to high-content screening. J Biomol Screen 15(7), 847–857.Google Scholar
Evron, T., Daigle, T.L. & Caron, M.G. (2012). GRK2: Multiple roles beyond G protein-coupled receptor desensitization. Trends Pharmacol Sci 33(3), 154–164.Google Scholar
Fenistein, D., Lenseigne, B., Christophe, T., Brodin, P. & Genovesio, A. (2008). A fast, fully automated cell segmentation algorithm for high-throughput and high-content screening. Cytometry A 73(10), 958–964.Google Scholar
Ferguson, S.S. & Caron, M.G. (2004). Green fluorescent protein-tagged beta-arrestin translocation as a measure of G protein-coupled receptor activation. Methods Mol Biol 237, 121–126.Google Scholar
Ferrandon, S., Feinstein, T.N., Castro, M., Wang, B., Bouley, R., Potts, J.T., Gardella, T.J. & Vilardaga, J.P. (2009). Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat Chem Biol 5(10), 734–742.Google Scholar
Fichter, K.M., Flajolet, M., Greengard, P. & Vu, T.Q. (2010). Kinetics of G-protein-coupled receptor endosomal trafficking pathways revealed by single quantum dots. Proc Natl Acad Sci USA 107(43), 18658–18663.Google Scholar
Ghosh, M., Peterson, Y.K., Lanier, S.M. & Smrcka, A.V. (2003). Receptor- and nucleotide exchange-independent mechanisms for promoting G protein subunit dissociation. J Biol Chem 278(37), 34747–34750.Google Scholar
Ghosh, R.N., Chen, Y.T., DeBiasio, R., DeBiasio, R.L., Conway, B.R., Minor, L.K. & Demarest, K.T. (2000). Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking. BioTechniques 29(1), 170–175.Google Scholar
Haasen, D., Schnapp, A., Valler, M.J. & Heilker, R. (2006). G protein-coupled receptor internalization assays in the high-content screening format. In Methods in Enzymology, James, I. (Ed.), pp. 121–139. New York: Academic Press.Google Scholar
Hamdan, F.F., Audet, M., Garneau, P., Pelletier, J. & 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(5), 463–475.Google Scholar
Hanyaloglu, A.C. & von Zastrow, M. (2008). Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu Rev Pharmacol Toxicol 48, 537–568.Google Scholar
Hudson, C.C., Oakley, R.H., Sjaastad, M.D. & Loomis, C.R. (2006). High-content screening of known G protein-coupled receptors by arrestin translocation. Methods Enzymol 414, 63–78.CrossRefGoogle ScholarPubMed
Kamal, M., Marquez, M., Vauthier, V., Leloire, A., Froguel, P., Jockers, R. & Couturier, C. (2009). Improved donor/acceptor BRET couples for monitoring beta-arrestin recruitment to G protein-coupled receptors. Biotechnol J 4(9), 1337–1344.Google Scholar
Kaya, A.I., Onaran, H.O., Ozcan, G., Ambrosio, C., Costa, T., Balli, S. & Ugur, O. (2012). Cell contact-dependent functional selectivity of beta2-adrenergic receptor ligands in stimulating cAMP accumulation and extracellular signal-regulated kinase phosphorylation. J Biol Chem 287(9), 6362–6374.Google Scholar
Kendall, R.T. & Luttrell, L.M. (2009). Diversity in arrestin function. Cell Mol Life Sci 66(18), 2953–2973.Google Scholar
Kwon, Y.J., Lee, W., Genovesio, A. & Emans, N. (2012). A high-content subtractive screen for selecting small molecules affecting internalization of GPCRs. J Biomol Screen 17(3), 379–385.CrossRefGoogle ScholarPubMed
Luttrell, L.M. & Lefkowitz, R.J. (2002). The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci 115(Pt 3), 455–465.CrossRefGoogle ScholarPubMed
Luttrell, L.M., Roudabush, F.L., Choy, E.W., Miller, W.E., Field, M.E., Pierce, K.L. & Lefkowitz, R.J. (2001). Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. Proc Natl Acad Sci USA 98(5), 2449–2454.Google Scholar
Magalhaes, A.C., Dunn, H. & Ferguson, S.S. (2011). Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins. Br J Pharmacol 165(6), 1717–1736.Google Scholar
McCaffrey, G., Welker, J., Scott, J., der Salm, L. & Grimes, M.L. (2009). High-resolution fractionation of signaling endosomes containing different receptors. Traffic (Copenhagen, Denmark) 10(7), 938–950.Google Scholar
Perroy, J., Pontier, S., Charest, P.G., Aubry, M. & Bouvier, M. (2004). Real-time monitoring of ubiquitination in living cells by BRET. Nat Methods 1(3), 203–208.Google Scholar
Peterson, Y.K., Bernard, M.L., Ma, H., Hazard, S. 3rd, Graber, S.G. & Lanier, S.M. (2000). Stabilization of the GDP-bound conformation of Gialpha by a peptide derived from the G-protein regulatory motif of AGS3. J Biol Chem 275(43), 33193–33196.Google Scholar
Ross, D.A., Lee, S., Reiser, V., Xue, J., Alves, K., Vaidya, S., Kreamer, A., Mull, R., Hudak, E., Hare, T., Detmers, P.A., Lingham, R., Ferrer, M., Strulovici, B. & Santini, F. (2008). Multiplexed assays by high-content imaging for assessment of GPCR activity. J Biomol Screen 13(6), 449–455.Google Scholar
Shenoy, S.K., Barak, L.S., Xiao, K., Ahn, S., Berthouze, M., Shukla, A.K., Luttrell, L.M. & Lefkowitz, R.J. (2007). Ubiquitination of beta-arrestin links seven-transmembrane receptor endocytosis and ERK activation. J Biol Chem 282(40), 29549–29562.Google Scholar
Stallaert, W., Dorn, J.F., van der Westhuizen, E., Audet, M. & Bouvier, M. (2012). Impedance responses reveal beta(2)-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles. PLoS One 7(1), e29420. Google Scholar
Tesmer, J.J. (2010). The quest to understand heterotrimeric G protein signaling. Nat Struct Molec Biol 17(6), 650–652.Google Scholar
Tohgo, A., Choy, E.W., Gesty-Palmer, D., Pierce, K.L., Laporte, S., Oakley, R.H., Caron, M.G., Lefkowitz, R.J. & Luttrell, L.M. (2003). The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. J Biol Chem 278(8), 6258–6267.Google Scholar
Vilardaga, J.P., Gardella, T.J., Wehbi, V.L. & Feinstein, T.N. (2012). Non-canonical signaling of the PTH receptor. Trends Pharmacol Sci 33(8), 423–431.Google Scholar
Vilardaga, J.P., Romero, G., Friedman, P.A. & Gardella, T.J. (2010). Molecular basis of parathyroid hormone receptor signaling and trafficking: A family B GPCR paradigm. Cell Mol Life Sci 68(1), 1–13.Google Scholar
Wills, L.P., Trager, R.E., Beeson, G.C., Lindsey, C.C., Peterson, Y.K., Beeson, C.C. & Schnellmann, R.G. (2012). The β2 adrenoceptor agonist formoterol stimulates mitochondrial biogenesis. J Pharmacol Exp Ther 342(1), 106–118.Google Scholar
Yu, N., Atienza, J.M., Bernard, J., Blanc, S., Zhu, J., Wang, X., Xu, X. & Abassi, Y.A. (2006). Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: An approach to study G protein-coupled receptors. Anal Chem 78(1), 35–43.Google Scholar
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