Generic placeholder image

Current Molecular Medicine

Editor-in-Chief

ISSN (Print): 1566-5240
ISSN (Online): 1875-5666

General Research Article

Single-cell Analysis of β2-Adrenergic Receptor Dynamics by Quantitative Fluorescence Microscopy

Author(s): Esraa Haji, Saeed Al Mahri, Yumna Aloraij, Shuja Malik and Sameer Mohammad*

Volume 20, Issue 6, 2020

Page: [488 - 493] Pages: 6

DOI: 10.2174/1566524020666191216125825

Price: $65

Abstract

Background: G protein-coupled receptors (GPCRs) represent the largest family of surface proteins and are involved in the regulation of key physiological processes. GPCRs are characterized by seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. Cellular response of these receptors to their ligands is largely determined by their surface expression and postactivation behavior including expression, desensitization and resensitization.

Objective: To develop a quantitative fluorescence Microscopy assay to study β2- Adrenergic receptor expression and desensitization.

Method: β2-Adrenergic receptor cDNA was engineered to put an HA tag at the extracellular N-terminus and GFP Tag at the intracellular C-terminus. GFP fluorescence serves as a measure of total cellular expression; whereas staining with CY3 conjugated anti-HA antibodies without permeabilizing the cells represents the surface expression of β2-AR. The images are quantified and amount of CY3 (surface) and GFP (total) fluorescence for each cell determined using image processing software.

Results: The method is sensitive and allows for the simultaneous measurement of surface and total expression of β2-AR.

Conclusion: A highly accurate method is described for measuring β2-AR surface and total expression based on single-cell quantitative immunofluorescence. The method can be used to determine agonist-induced desensitization and resensitization process as well as receptor kinetics like endocytosis and exocytosis of β2-Adrenergic receptor and can be applied to essentially any other GPCR.

Keywords: G-protein coupled receptors, receptor desensitization, quantitative immune-flourescence, fluorescence microscopy, C-terminus, microscopy assay.

[1]
Ribas C, Penela P, Murga C, et al. The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling. Biochim Biophys Acta 2007; 1768(4): 913-22.
[http://dx.doi.org/10.1016/j.bbamem.2006.09.019] [PMID: 17084806]
[2]
Salazar NC, Chen J, Rockman HA. Cardiac GPCRs: GPCR signaling in healthy and failing hearts. Biochim Biophys Acta 2007; 1768(4): 1006-18.
[http://dx.doi.org/10.1016/j.bbamem.2007.02.010] [PMID: 17376402]
[3]
Williams C, Hill SJ. GPCR signaling: understanding the pathway to successful drug discovery. Methods Mol Biol 2009; 552: 39-50.
[http://dx.doi.org/10.1007/978-1-60327-317-6_3] [PMID: 19513640]
[4]
Borroto-Escuela DO, Carlsson J, Ambrogini P, et al. Understanding the role of GPCR Heteroreceptor complexes in modulating the brain networks in health and disease. Front Cell Neurosci 2017; 11: 37.
[http://dx.doi.org/10.3389/fncel.2017.00037] [PMID: 28270751]
[5]
Gurevich VV, Gurevich EV. GPCR signaling regulation: the role of grks and arrestins. Front Pharmacol 2019; 10: 125.
[http://dx.doi.org/10.3389/fphar.2019.00125] [PMID: 30837883]
[6]
Harris DM, Cohn HI, Pesant S, Eckhart AD. GPCR signalling in hypertension: role of GRKs. Clin Sci (Lond) 2008; 115(3): 79-89.
[http://dx.doi.org/10.1042/CS20070442] [PMID: 18593382]
[7]
Nebigil CG, Désaubry L. The role of GPCR signaling in cardiac epithelial to mesenchymal transformation (EMT). Trends Cardiovasc Med 2019; 29(4): 200-4.
[http://dx.doi.org/10.1016/j.tcm.2018.08.007] [PMID: 30172578]
[8]
Wang D. The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity. Immunopharmacol Immunotoxicol 2018; 40(3): 187-92.
[http://dx.doi.org/10.1080/08923973.2018.1434792] [PMID: 29433403]
[9]
Mohammad S. GPR40 Agonists for the treatment of type 2 diabetes mellitus: benefits and challenges. Curr Drug Targets 2016; 17(11): 1292-300.
[http://dx.doi.org/10.2174/1389450117666151209122702] [PMID: 26648068]
[10]
Mohammad S. Role of free fatty acid receptor 2 (FFAR2) in the regulation of metabolic homeostasis. Curr Drug Targets 2015; 16(7): 771-5.
[http://dx.doi.org/10.2174/1389450116666150408103557] [PMID: 25850624]
[11]
Mohammad S, Ramos LS, Buck J, Levin LR, Rubino F, McGraw TE. Gastric inhibitory peptide controls adipose insulin sensitivity via activation of cAMP-response element-binding protein and p110β isoform of phosphatidylinositol 3-kinase. J Biol Chem 2011; 286(50): 43062-70.
[http://dx.doi.org/10.1074/jbc.M111.289009] [PMID: 22027830]
[12]
Stoddart LA, Kilpatrick LE, Briddon SJ, Hill SJ. Probing the pharmacology of G protein-coupled receptors with fluorescent ligands. Neuropharmacology 2015; 98: 48-57.
[http://dx.doi.org/10.1016/j.neuropharm.2015.04.033] [PMID: 25979488]
[13]
Kypreos M, Banerjee T, Mukherjee D. G protein-coupled receptors--potential roles in clinical pharmacology. Cardiovasc Hematol Agents Med Chem 2014; 12(1): 29-33.
[http://dx.doi.org/10.2174/187152571201141201093751] [PMID: 25470151]
[14]
Solinski HJ, Gudermann T, Breit A. Pharmacology and signaling of MAS-related G protein-coupled receptors. Pharmacol Rev 2014; 66(3): 570-97.
[http://dx.doi.org/10.1124/pr.113.008425] [PMID: 24867890]
[15]
Summers RJ. Molecular pharmacology of G protein-coupled receptors. Editorial Br J Pharmacol 2010; 159(5): 983-5. [Editorial].
[http://dx.doi.org/10.1111/j.1476-5381.2010.00695.x] [PMID: 20388130]
[16]
Gilchrist A. Modulating G-protein-coupled receptors: from traditional pharmacology to allosterics. Trends Pharmacol Sci 2007; 28(8): 431-7.
[http://dx.doi.org/10.1016/j.tips.2007.06.012] [PMID: 17644194]
[17]
Abbracchio MP, Burnstock G, Boeynaems JM, et al. International union of pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 2006; 58(3): 281-341.
[http://dx.doi.org/10.1124/pr.58.3.3] [PMID: 16968944]
[18]
Granell S, Mohammad S, Ramanagoudr-Bhojappa R, Baldini G. Obesity-linked variants of melanocortin-4 receptor are misfolded in the endoplasmic reticulum and can be rescued to the cell surface by a chemical chaperone. Mol Endocrinol 2010; 24(9): 1805-21.
[http://dx.doi.org/10.1210/me.2010-0071] [PMID: 20631012]
[19]
Mohammad S, Baldini G, Granell S, Narducci P, Martelli AM, Baldini G. Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons. J Biol Chem 2007; 282(7): 4963-74.
[http://dx.doi.org/10.1074/jbc.M608283200] [PMID: 17166828]
[20]
Lobingier BT, von Zastrow M. When trafficking and signaling mix: How subcellular location shapes G protein-coupled receptor activation of heterotrimeric G proteins. Traffic 2019; 20(2): 130-6.
[http://dx.doi.org/10.1111/tra.12634] [PMID: 30578610]
[21]
Kang DS, Tian X, Benovic JL. Role of β-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking. Curr Opin Cell Biol 2014; 27: 63-71.
[http://dx.doi.org/10.1016/j.ceb.2013.11.005] [PMID: 24680432]
[22]
Margus H, Padari K, Pooga M. Insights into cell entry and intracellular trafficking of peptide and protein drugs provided by electron microscopy. Adv Drug Deliv Rev 2013; 65(8): 1031-8.
[http://dx.doi.org/10.1016/j.addr.2013.04.013] [PMID: 23624037]
[23]
Enns C. Overview of protein trafficking in the secretory and endocytic pathways. In: urr Protoc Cell Biol. 2001.
[24]
Xu ZQ, Zhang X, Scott L. Regulation of G protein-coupled receptor trafficking. Acta Physiol (Oxf) 2007; 190(1): 39-45.
[http://dx.doi.org/10.1111/j.1365-201X.2007.01695.x] [PMID: 17428231]
[25]
Schwartz AL. Cell biology of intracellular protein trafficking. Annu Rev Immunol 1990; 8: 195-229.
[http://dx.doi.org/10.1146/annurev.iy.08.040190.001211] [PMID: 2160830]
[26]
Kumagai H, Ikeda Y, Motozawa Y, et al. Quantitative measurement of GPCR endocytosis via pulse-chase covalent labeling. PLoS One 2015; 10(5)e0129394
[http://dx.doi.org/10.1371/journal.pone.0129394] [PMID: 26020647]
[27]
Navratilova I, Besnard J, Hopkins AL. Screening for GPCR ligands using surface plasmon resonance. ACS Med Chem Lett 2011; 2(7): 549-54.
[http://dx.doi.org/10.1021/ml2000017] [PMID: 21765967]
[28]
Hislop JN, von Zastrow M. Analysis of GPCR localization and trafficking. Methods Mol Biol 2011; 746: 425-40.
[http://dx.doi.org/10.1007/978-1-61779-126-0_25] [PMID: 21607873]
[29]
Xu X, Brzostowski JA, Jin T. Monitoring dynamic GPCR signaling events using fluorescence microscopy, FRET imaging, and single-molecule imaging. Methods Mol Biol 2009; 571: 371-83.
[http://dx.doi.org/10.1007/978-1-60761-198-1_25] [PMID: 19763980]
[30]
Sorkin A, Duex JE. Quantitative analysis of endocytosis and turnover of epidermal growth factor (EGF) and EGF receptor In: Curr Protoc Cell Biol. 2010.
[31]
Gabriel L, Stevens Z, Melikian H. Measuring plasma membrane protein endocytic rates by reversible biotinylation. J Vis Exp 2009; 2009(34)e1669
[http://dx.doi.org/10.3791/1669]
[32]
Nevins AM, Marchese A. Detecting cell surface expression of the g protein-coupled receptor CXCR4. Methods Mol Biol 2018; 1722: 151-64.
[http://dx.doi.org/10.1007/978-1-4939-7553-2_10] [PMID: 29264804]
[33]
Kelly E, Bailey CP, Henderson G. Agonist-selective mechanisms of GPCR desensitization. Br J Pharmacol 2008; 153(Suppl. 1): S379-88.
[http://dx.doi.org/10.1038/sj.bjp.0707604] [PMID: 18059321]
[34]
Pennock RL, Hentges ST. Desensitization-resistant and -sensitive GPCR-mediated inhibition of GABA release occurs by Ca2+-dependent and -independent mechanisms at a hypothalamic synapse. J Neurophysiol 2016; 115(5): 2376-88.
[http://dx.doi.org/10.1152/jn.00535.2015] [PMID: 26912590]
[35]
Rajagopal S, Shenoy SK. GPCR desensitization: Acute and prolonged phases. Cell Signal 2018; 41: 9-16.
[http://dx.doi.org/10.1016/j.cellsig.2017.01.024] [PMID: 28137506]
[36]
Charlton SJ. Agonist efficacy and receptor desensitization: from partial truths to a fuller picture. Br J Pharmacol 2009; 158(1): 165-8.
[http://dx.doi.org/10.1111/j.1476-5381.2009.00352.x] [PMID: 19719779]
[37]
Kohout TA, Lefkowitz RJ. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol 2003; 63(1): 9-18.
[http://dx.doi.org/10.1124/mol.63.1.9] [PMID: 12488531]
[38]
Mohammad S, Patel RT, Bruno J, Panhwar MS, Wen J, McGraw TE. A naturally occurring GIP receptor variant undergoes enhanced agonist-induced desensitization, which impairs GIP control of adipose insulin sensitivity. Mol Cell Biol 2014; 34(19): 3618-29.
[http://dx.doi.org/10.1128/MCB.00256-14] [PMID: 25047836]
[39]
Halls ML, Canals M. Genetically encoded FRET biosensors to illuminate compartmentalised GPCR signalling. Trends Pharmacol Sci 2018; 39(2): 148-57.
[http://dx.doi.org/10.1016/j.tips.2017.09.005] [PMID: 29054309]
[40]
Hein L, Ishii K, Coughlin SR, Kobilka BK. Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. J Biol Chem 1994; 269(44): 27719-26.
[PMID: 7961693]
[41]
Ho TT, Nguyen JT, Liu J, et al. Method for rapid optimization of recombinant GPCR protein expression and stability using virus-like particles. Protein Expr Purif 2017; 133: 41-9.
[http://dx.doi.org/10.1016/j.pep.2017.03.002] [PMID: 28263854]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy