Login Register Cart 0
Generic placeholder image

Current Pharmaceutical Design

Editor-in-Chief

ISSN (Print): 1381-6128
ISSN (Online): 1873-4286

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Review Article

Fluorescence-based Methods for the Study of Protein-Protein Interactions Modulated by Ligand Binding

Author(s): Michael R. Stoneman, Naomi Raicu, Gabriel Biener and Valerică Raicu*

Volume 26, Issue 44, 2020

Page: [5668 - 5683] Pages: 16

DOI: 10.2174/1381612826666201116120934

Price: $65

Abstract

Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomers but that the oligomers also may modulate the receptor function provides a promising avenue in the area of drug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacy while reducing side effects. In this regard, determining the various oligomeric configurations and geometric sub-states of a membrane receptor is of utmost importance.

Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring the quaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescence intensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiency are analyzed using theoretical models of various quaternary structures to determine the geometry and stoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensities are analyzed to reveal quantitative information on the size and stability of protein oligomers.

Results: We demonstrate the application of these techniques to a number of different fluorescence-based studies of cells expressing fluorescently labeled membrane receptors, both in the presence and absence of various ligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regarding the quaternary structure receptors form, as well as FIF and FRET for determining the relative abundance of different-sized oligomers when an equilibrium forms between such structures.

Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers, which are of great benefit to structure‐based drug design.

Keywords: FRET, fluorescence fluctuation spectroscopy, protein-protein interactions, FRET spectrometry, fluorescence imaging, ligand effect, oligomerization, quaternary structure.

« Previous Next »
[1]
Rosenbaum DM, Rasmussen SG, Kobilka BK. The structure and function of G-protein-coupled receptors. Nature 2009; 459(7245): 356-63.
[http://dx.doi.org/10.1038/nature08144] [PMID: 19458711]
[2]
Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM. Molecular signatures of G-protein-coupled receptors. Nature 2013; 494(7436): 185-94.
[http://dx.doi.org/10.1038/nature11896 ] [PMID: 23407534]
[3]
Milligan G. The prevalence, maintenance, and relevance of G protein-coupled receptor oligomerization. Mol Pharmacol 2013; 84(1): 158-69.
[http://dx.doi.org/10.1124/mol.113.084780] [PMID: 23632086]
[4]
Lagerström MC, Schiöth HB. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 2008; 7(4): 339-57.
[http://dx.doi.org/10.1038/nrd2518] [PMID: 18382464]
[5]
Palczewski K. Oligomeric forms of G protein-coupled receptors (GPCRs). Trends Biochem Sci 2010; 35(11): 595-600.
[http://dx.doi.org/10.1016/j.tibs.2010.05.002] [PMID: 20538466]
[6]
Farran B. An update on the physiological and therapeutic relevance of GPCR oligomers. Pharmacol Res 2017; 117: 303-27.
[http://dx.doi.org/10.1016/j.phrs.2017.01.008] [PMID: 28087443]
[7]
Park PSH, Wells JW. Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells Biochemistry 2003; 42(44): 12960-71.
[http://dx.doi.org/10.1021/bi034491m] [PMID: 14596611]
[8]
Milligan G. G protein-coupled receptor hetero-dimerization: contribution to pharmacology and function. Br J Pharmacol 2009; 158(1): 5-14.
[http://dx.doi.org/10.1111/j.1476-5381.2009.00169.x] [PMID: 19309353]
[9]
Raicu V, Stoneman MR, Fung R, et al. Determination of supramolecular structure and spatial distribution of protein complexes in living cells. Nat Photonics 2009; 3: 107-13.
[http://dx.doi.org/10.1038/nphoton.2008.291]
[10]
Ferré S. The GPCR heterotetramer: challenging classical pharmacology. Trends Pharmacol Sci 2015; 36(3): 145-52.
[http://dx.doi.org/10.1016/j.tips.2015.01.002] [PMID: 25704194]
[11]
Overton MC, Chinault SL, Blumer KJ. Oligomerization of G-protein-coupled receptors: lessons from the yeast Saccharomyces cerevisiae. Eukaryot Cell 2005; 4(12): 1963-70.
[http://dx.doi.org/10.1128/EC.4.12.1963-1970.2005] [PMID: 16339714]
[12]
Maurel D, Comps-Agrar L, Brock C, et al. Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat Methods 2008; 5(6): 561-7.
[http://dx.doi.org/10.1038/nmeth.1213] [PMID: 18488035]
[13]
Lundstrom K. An overview on GPCRs and drug discovery: structure-based drug design and structural biology on GPCRs. Methods Mol Biol 2009; 552: 51-66.
[http://dx.doi.org/10.1007/978-1-60327-317-6_4] [PMID: 19513641]
[14]
Anderson AC. The process of structure-based drug design. Chem Biol 2003; 10(9): 787-97.
[http://dx.doi.org/10.1016/j.chembiol.2003.09.002] [PMID: 14522049]
[15]
Dalrymple MB, Pfleger KDG, Eidne KA. G protein-coupled receptor dimers: functional consequences, disease states and drug targets. Pharmacol Ther 2008; 118(3): 359-71.
[http://dx.doi.org/10.1016/j.pharmthera.2008.03.004] [PMID: 18486226]
[16]
Panetta R, Greenwood MT. Physiological relevance of GPCR oligomerization and its impact on drug discovery. Drug Discov Today 2008; 13(23-24): 1059-66.
[http://dx.doi.org/10.1016/j.drudis.2008.09.002] [PMID: 18824244]
[17]
Glass M, Govindpani K, Furkert DP, Hurst DP, Reggio PH, Flanagan JU. One for the price of two…are bivalent ligands targeting cannabinoid receptor dimers capable of simultaneously binding to both receptors? Trends Pharmacol Sci 2016; 37(5): 353-63.
[http://dx.doi.org/10.1016/j.tips.2016.01.010] [PMID: 26917061]
[18]
Carli M, Kolachalam S, Aringhieri S, et al. Dopamine D2 receptors dimers: how can we pharmacologically target them? Curr Neuropharmacol 2018; 16(2): 222-30.
[http://dx.doi.org/10.2174/1570159X15666170518151127] [PMID: 28521704]
[19]
Stoneman MR, Biener G, Ward RJ, et al. A general method to quantify ligand-driven oligomerization from fluorescence-based images. Nat Methods 2019; 16(6): 493-6.
[http://dx.doi.org/10.1038/s41592-019-0408-9] [PMID: 31110281]
[20]
Milligan G, Ward RJ, Marsango S. GPCR homo-oligomerization. Curr Opin Cell Biol 2019; 57: 40-7.
[http://dx.doi.org/10.1016/j.ceb.2018.10.007] [PMID: 30453145]
[21]
Raicu V, Singh DR. FRET spectrometry: a new tool for the determination of protein quaternary structure in living cells. Biophys J 2013; 105(9): 1937-45.
[http://dx.doi.org/10.1016/j.bpj.2013.09.015] [PMID: 24209838]
[22]
Selvin PR. The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 2000; 7(9): 730-4.
[http://dx.doi.org/10.1038/78948] [PMID: 10966639]
[23]
Förster T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 1948; 2: 55-75.
[http://dx.doi.org/10.1002/andp.19484370105]
[24]
Clegg RM. Fluorescence resonance energy transfer Fluorescence Imaging Spectroscopy and Microscopy New York: Wiley 1996; 179-252.
[25]
Chen L, Novicky L, Merzlyakov M, Hristov T, Hristova K. Measuring the energetics of membrane protein dimerization in mammalian membranes. J Am Chem Soc 2010; 132(10): 3628-35.
[http://dx.doi.org/10.1021/ja910692u] [PMID: 20158179]
[26]
Singh DR, Kanvinde P, King C, Pasquale EB, Hristova K. The EphA2 receptor is activated through induction of distinct, ligand-dependent oligomeric structures. Commun Biol 2018; 1: 1-12.
[http://dx.doi.org/10.1038/s42003-018-0017-7]
[27]
Albizu L, Cottet M, Kralikova M, et al. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol 2010; 6(8): 587-94.
[http://dx.doi.org/10.1038/nchembio.396 ] [PMID: 20622858]
[28]
King C, Wirth D, Workman S, Hristova K. Interactions between NRP1 and VEGFR2 molecules in the plasma membrane. Biochim Biophys Acta Biomembr 2018; 1860(10): 2118-25.
[http://dx.doi.org/10.1016/j.bbamem.2018.03.023] [PMID: 29630862]
[29]
King C, Hristova K. Direct measurements of VEGF-VEGFR2 binding affinities reveal the coupling between ligand binding and receptor dimerization. J Biol Chem 2019; 294(23): 9064-75.
[http://dx.doi.org/10.1074/jbc.RA119.007737] [PMID: 31023826]
[30]
Mishra AK, Gragg M, Stoneman MR, et al. Quaternary structures of opsin in live cells revealed by FRET spectrometry. Biochem J 2016; 473(21): 3819-36.
[http://dx.doi.org/10.1042/BCJ20160422] [PMID: 27623775]
[31]
Schmidt S, Jakab M, Costa I, et al. Quaternary structure assessment of ICln by fluorescence resonance energy transfer (FRET) in vivo. Cell Physiol Biochem 2009; 23(4-6): 397-406.
[http://dx.doi.org/10.1159/000218186] [PMID: 19471107]
[32]
Demarco IA, Periasamy A, Booker CF, Day RN. Monitoring dynamic protein interactions with photoquenching FRET. Nat Methods 2006; 3(7): 519-24.
[http://dx.doi.org/10.1038/nmeth889] [PMID: 16791209]
[33]
Ferrari ML, Gomez GA, Maccioni HJF. Spatial organization and stoichiometry of N-terminal domain-mediated glycosyltransferase complexes in Golgi membranes determined by fret microscopy. Neurochem Res 2012; 37(6): 1325-34.
[http://dx.doi.org/10.1007/s11064-012-0741-1] [PMID: 22388569]
[34]
Hochreiter B, Kunze M, Moser B, Schmid JA. Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells. Sci Rep 2019; 9(1): 8233.
[http://dx.doi.org/10.1038/s41598-019-44650-0] [PMID: 31160659]
[35]
Stoneman MR, Paprocki JD, Biener G, et al. Quaternary structure of the yeast pheromone receptor Ste2 in living cells. Biochim Biophys Acta Biomembr 2017; 1859(9 Pt A): 1456-64.
[http://dx.doi.org/10.1016/j.bbamem.2016.12.008] [PMID: 27993568]
[36]
Li M, Reddy LG, Bennett R, Silva ND Jr, Jones LR, Thomas DD. A fluorescence energy transfer method for analyzing protein oligomeric structure: application to phospholamban. Biophys J 1999; 76(5): 2587-99.
[http://dx.doi.org/10.1016/S0006-3495(99)77411-4] [PMID: 10233073]
[37]
Woehler A, Wlodarczyk J, Ponimaskin EG. Specific oligomerization of the 5-HT1A receptor in the plasma membrane. Glycoconj J 2009; 26(6): 749-56.
[http://dx.doi.org/10.1007/s10719-008-9187-8] [PMID: 18853255]
[38]
King C, Raicu V, Hristova K. Understanding the FRET signatures of interacting membrane proteins. J Biol Chem 2017; 292(13): 5291-310.
[http://dx.doi.org/10.1074/jbc.M116.764282] [PMID: 28188294]
[39]
Raicu V. Ab Initio Derivation of the FRET equations resolves old puzzles and suggests measurement strategies. Biophys J 2019; 116(7): 1313-27.
[http://dx.doi.org/10.1016/j.bpj.2019.02.016 ] [PMID: 30885378]
[40]
Raicu V. Efficiency of resonance energy transfer in homo-oligomeric complexes of proteins. J Biol Phys 2007; 33(2): 109-27.
[http://dx.doi.org/10.1007/s10867-007-9046-z] [PMID: 19669544]
[41]
Singh DR, Mohammad MM, Patowary S, et al. Determination of the quaternary structure of a bacterial ATP-binding cassette (ABC) transporter in living cells. Integr Biol 2013; 5(2): 312-23.
[http://dx.doi.org/10.1039/c2ib20218b] [PMID: 23223798]
[42]
Harikumar KG, Miller LJ. Monitoring the state of cholecystokinin receptor oligomerization after ligand binding using decay of time-resolved fluorescence anisotropy. Ann N Y Acad Sci 2008; 1144: 21-7.
[http://dx.doi.org/10.1196/annals.1418.004] [PMID: 19076359]
[43]
Ward RJ, Pediani JD, Godin AG, Milligan G. Regulation of oligomeric organization of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor observed by spatial intensity distribution analysis. J Biol Chem 2015; 290(20): 12844-57.
[http://dx.doi.org/10.1074/jbc.M115.644724] [PMID: 25825490]
[44]
Godin AG, Costantino S, Lorenzo LE, et al. Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis. Proc Natl Acad Sci USA 2011; 108(17): 7010-5.
[http://dx.doi.org/10.1073/pnas.1018658108] [PMID: 21482753]
[45]
Sinclair MB, Haaland DM, Timlin JA, Jones HDT. Hyperspectral confocal microscope. Appl Opt 2006; 45(24): 6283-91.
[http://dx.doi.org/10.1364/AO.45.006283] [PMID: 16892134]
[46]
Zhang Z, Kenny SJ, Hauser M, Li W, Xu K. Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nat Methods 2015; 12(10): 935-8.
[http://dx.doi.org/10.1038/nmeth.3528] [PMID: 26280329]
[47]
Biener G, Stoneman MR, Acbas G, et al. Development and experimental testing of an optical micro-spectroscopic technique incorporating true line-scan excitation. Int J Mol Sci 2013; 15(1): 261-76.
[http://dx.doi.org/10.3390/ijms15010261] [PMID: 24378851]
[48]
Elliott AD, Gao L, Ustione A, et al. Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer. J Cell Sci 2012; 125(Pt 20): 4833-40.
[http://dx.doi.org/10.1242/jcs.108258] [PMID: 22854044]
[49]
Lavagnino Z, Dwight J, Ustione A, Nguyen TU, Tkaczyk TS, Piston DW. Snapshot hyperspectral light-sheet imaging of signal transduction in live pancreatic islets. Biophys J 2016; 111(2): 409-17.
[http://dx.doi.org/10.1016/j.bpj.2016.06.014] [PMID: 27463142]
[50]
Neher R, Neher E. Optimizing imaging parameters for the separation of multiple labels in a fluorescence image. J Microsc 2004; 213(1): 46-62.
[http://dx.doi.org/10.1111/j.1365-2818.2004.01262.x ] [PMID: 14678512]
[51]
Thaler C, Koushik SV, Blank PS, Vogel SS. Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophys J 2005; 89(4): 2736-49.
[http://dx.doi.org/10.1529/biophysj.105.061853] [PMID: 16040744]
[52]
Zimmermann T, Rietdorf J, Girod A, Georget V, Pepperkok R. Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett 2002; 531(2): 245-9.
[http://dx.doi.org/10.1016/S0014-5793(02)03508-1] [PMID: 12417320]
[53]
Mishra AK, Mavlyutov T, Singh DR, et al. The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands. Biochem J 2015; 466(2): 263-71.
[http://dx.doi.org/10.1042/BJ20141321] [PMID: 25510962]
[54]
Chen Y, Müller JD, So PT, Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 1999; 77(1): 553-67.
[http://dx.doi.org/10.1016/S0006-3495(99)76912-2] [PMID: 10388780]
[55]
Herrick-Davis K, Grinde E, Cowan A, Mazurkiewicz JE. Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle. Mol Pharmacol 2013; 84(4): 630-42.
[http://dx.doi.org/10.1124/mol.113.087072] [PMID: 23907214]
[56]
Digman MA, Dalal R, Horwitz AF, Gratton E. Mapping the number of molecules and brightness in the laser scanning microscope. Biophys J 2008; 94(6): 2320-32.
[http://dx.doi.org/10.1529/biophysj.107.114645] [PMID: 18096627]
[57]
Unruh JR, Gratton E. Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera. Biophys J 2008; 95(11): 5385-98.
[http://dx.doi.org/10.1529/biophysj.108.130310] [PMID: 18805922]
[58]
Nagy P, Claus J, Jovin TM, Arndt-Jovin DJ. Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis. Proc Natl Acad Sci USA 2010; 107(38): 16524-9.
[http://dx.doi.org/10.1073/pnas.1002642107] [PMID: 20813958]
[59]
Pediani JD, Ward RJ, Marsango S, Milligan G. Spatial intensity distribution analysis: studies of G protein-coupled receptor oligomerisation. Trends Pharmacol Sci 2018; 39(2): 175-86.
[http://dx.doi.org/10.1016/j.tips.2017.09.001] [PMID: 29032835]
[60]
Digman MA, Wiseman PW, Choi C, Horwitz AR, Gratton E. Stoichiometry of molecular complexes at adhesions in living cells. Proc Natl Acad Sci USA 2009; 106(7): 2170-5.
[http://dx.doi.org/10.1073/pnas.0806036106] [PMID: 19168634]
[61]
Godin AG, Rappaz B, Potvin-Trottier L, Kennedy TE, De Koninck Y, Wiseman PW. Spatial intensity distribution analysis reveals abnormal oligomerization of proteins in single cells. Biophys J 2015; 109(4): 710-21.
[http://dx.doi.org/10.1016/j.bpj.2015.06.068] [PMID: 26287623]
[62]
Chen Y, Wei LN, Müller JD. Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Proc Natl Acad Sci USA 2003; 100(26): 15492-7.
[http://dx.doi.org/10.1073/pnas.2533045100] [PMID: 14673112]
[63]
Stoneman MR, Biener G, Raicu V. Reply to: Spatial heterogeneity in molecular brightness. Nat Methods 2020; 17(3): 276-8.
[http://dx.doi.org/10.1038/s41592-020-0735-x] [PMID: 32042189]
[64]
Achanta R, Shaji A, Smith K, Lucchi A, Fua P, Süsstrunk S. SLIC superpixels compared to state-of-the-art superpixel methods. IEEE Trans Pattern Anal Mach Intell 2012; 34(11): 2274-82.
[http://dx.doi.org/10.1109/TPAMI.2012.120] [PMID: 22641706]
[65]
Achanta R, Shaji A, Smith K, Luchhi A, Fua P, Susstrunk S. SLIC Superpixels EPFL Technical Report 2010; 149300 1 -15
[66]
Zhang YX, Li XM, Gao XF, Zhang CM. A simple algorithm of superpixel segmentation with boundary constraint. IEEE Trans Circ Syst Video Tech 2017; 27: 1502-14.
[67]
Miller LJ, Dong M, Harikumar KG. Ligand binding and activation of the secretin receptor, a prototypic family B G protein-coupled receptor. Br J Pharmacol 2012; 166(1): 18-26.
[http://dx.doi.org/10.1111/j.1476-5381.2011.01463.x] [PMID: 21542831]
[68]
Raicu V. Extraction of information on macromolecular interactions from fluorescence micro-spectroscopy measurements in the presence and absence of FRET. Spectrochim Acta A Mol Biomol Spectrosc 2018; 199: 340-8.
[http://dx.doi.org/10.1016/j.saa.2018.03.075] [PMID: 29631099]
[69]
Stoneman MR, Biener G, Raicu V. Proposal for simultaneous analysis of fluorescence intensity fluctuations and resonance energy transfer (IFRET) measurements. Methods Appl Fluoresc 2020; 8(3)035011
[http://dx.doi.org/10.1088/2050-6120/ab9b68] [PMID: 32521525]

Rights & Permissions Print Cite
Article Metrics
25
World Health Day
© 2024 Bentham Science Publishers | Privacy Policy