Overcoming the Challenges of Detecting GPCR Oligomerization in the
Brain

Víctor       Fernández-Dueñas; Jordi       Bonaventura; Ester       Aso; Rafael       Luján; Sergi       Ferré; Francisco      Ciruela
Abstract

G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A_2A receptor (A₂AR)-dopamine D₂ receptor (D₂R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders.
Keywords: GPCR oligomerization, immunoelectron microscopy, proximity ligation assay, TR-FRET, ALPHA assay, RET.
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Graphical Abstract

[1] 
Fredriksson, R.; Lagerström, M.C.; Lundin, L.G.; Schiöth, H.B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol.,  2003, 63(6), 1256-1272.
[http://dx.doi.org/10.1124/mol.63.6.1256] [PMID:  12761335] 
[2] 
Attwood, T.K.; Findlay, J.B.C. Fingerprinting G-protein-coupled receptors. Protein Eng.,  1994, 7(2), 195-203.
[http://dx.doi.org/10.1093/protein/7.2.195] [PMID:  8170923] 
[3] 
Schiöth, H.B.; Fredriksson, R. The GRAFS classification system of G-protein coupled receptors in comparative perspective. Gen. Comp. Endocrinol.,  2005, 142(1-2), 94-101.
[http://dx.doi.org/10.1016/j.ygcen.2004.12.018] [PMID:  15862553] 
[4] 
Lagerström, M.C.; Schiöth, H.B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov.,  2008, 7(4), 339-357.
[http://dx.doi.org/10.1038/nrd2518] [PMID:  18382464] 
[5] 
Hu, G.M.; Mai, T.L.; Chen, C.M. Visualizing the GPCR network: classification and evolution. Sci. Rep.,  2017, 7(1), 15495-15510.
[http://dx.doi.org/10.1038/s41598-017-15707-9] [PMID:  29138525] 
[6] 
Erlandson, S.C.; McMahon, C.; Kruse, A.C. Structural basis for G protein-coupled receptor signaling. Annu. Rev. Biophys.,  2018, 47, 1-18.
[http://dx.doi.org/10.1146/annurev-biophys-070317-032931] [PMID:  29498889] 
[7] 
Weis, W.I.; Kobilka, B.K. The molecular basis of G protein-coupled receptor activation. Annu. Rev. Biochem.,  2018, 87, 897-919.
[http://dx.doi.org/10.1146/annurev-biochem-060614-033910] [PMID:  29925258] 
[8] 
Gurevich, V.V.; Gurevich, E.V. The structural basis of the arrestin binding to GPCRs. Mol. Cell. Endocrinol.,  2019, 484, 34-41.
[http://dx.doi.org/10.1016/j.mce.2019.01.019] [PMID:  30703488] 
[9] 
Urban, J.D.; Clarke, W.P.; von Zastrow, M.; Nichols, D.E.; Kobilka, B.; Weinstein, H.; Javitch, J.A.; Roth, B.L.; Christopoulos, A.; Sexton, P.M.; Miller, K.J.; Spedding, M.; Mailman, R.B. Functional selectivity and classical concepts of quantitative pharmacology. J. Pharmacol. Exp. Ther.,  2007, 320(1), 1-13.
[http://dx.doi.org/10.1124/jpet.106.104463] [PMID:  16803859] 
[10] 
Rozenfeld, R.; Devi, L.A. Receptor heterodimerization leads to a switch in signaling: beta-arrestin2-mediated ERK activation by mu-delta opioid receptor heterodimers. FASEB J.,  2007, 21(10), 2455-2465.
[http://dx.doi.org/10.1096/fj.06-7793com] [PMID:  17384143] 
[11] 
Guitart, X.; Navarro, G.; Moreno, E.; Yano, H.; Cai, N-S.; Sánchez-Soto, M.; Kumar-Barodia, S.; Naidu, Y.T.; Mallol, J.; Cortés, A.; Lluís, C.; Canela, E.I.; Casadó, V.; McCormick, P.J.; Ferré, S. Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer. Mol. Pharmacol.,  2014, 86(4), 417-429.
[http://dx.doi.org/10.1124/mol.114.093096] [PMID:  25097189] 
[12] 
Kenakin, T.; Miller, L.J. Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery. Pharmacol. Rev.,  2010, 62(2), 265-304.
[http://dx.doi.org/10.1124/pr.108.000992] [PMID:  20392808] 
[13] 
Milligan, G.; Ward, R.J.; Marsango, S. GPCR homo-oligomerization. Curr. Opin. Cell Biol.,  2019, 57, 40-47.
[http://dx.doi.org/10.1016/j.ceb.2018.10.007] [PMID:  30453145] 
[14] 
Ferré, S.; Casadó, V.; Devi, L.A.; Filizola, M.; Jockers, R.; Lohse, M.J.; Milligan, G.; Pin, J.P.; Guitart, X. G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives. Pharmacol. Rev.,  2014, 66(2), 413-434.
[http://dx.doi.org/10.1124/pr.113.008052] [PMID:  24515647] 
[15] 
Guidolin, D.; Marcoli, M.; Tortorella, C.; Maura, G.; Agnati, L.F. G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication. Rev. Neurosci.,  2018, 29(7), 703-726.
[http://dx.doi.org/10.1515/revneuro-2017-0087] [PMID:  29466243] 
[16] 
Agnati, L.F.; Fuxe, K.; Zoli, M.; Rondanini, C.; Ogren, S.O. New vistas on synaptic plasticity: the receptor mosaic hypothesis of the engram. Med. Biol.,  1982, 60(4), 183-190.
[PMID:  6128444] 
[17] 
Fuxe, K.; Agnati, L.F.; Benfenati, F.; Celani, M.; Zini, I.; Zoli, M.; Mutt, V. Evidence for the existence of receptor-receptor interactions in the central nervous system. Studies on the regulation of monoamine receptors by neuropeptides. J. Neural Transm. Suppl.,  1983, 18, 165-179.
[PMID:  6192208] 
[18] 
Fuxe, K.; Canals, M.; Torvinen, M.; Marcellino, D.; Terasmaa, A.; Genedani, S.; Leo, G.; Guidolin, D.; Diaz-Cabiale, Z.; Rivera, A.; Lundstrom, L.; Langel, U.; Narvaez, J.; Tanganelli, S.; Lluis, C.; Ferré, S.; Woods, A.; Franco, R.; Agnati, L.F. Intramembrane receptor-receptor interactions: a novel principle in molecular medicine. J. Neural Transm. (Vienna),  2007, 114(1), 49-75.
[http://dx.doi.org/10.1007/s00702-006-0589-0] [PMID:  17066251] 
[19] 
Ng, G.Y.K.; O’Dowd, B.F.; Lee, S.P.; Chung, H.T.; Brann, M.R.; Seeman, P.; George, S.R. Dopamine D2 receptor dimers and receptor-blocking peptides. Biochem. Biophys. Res. Commun.,  1996, 227(1), 200-204.
[http://dx.doi.org/10.1006/bbrc.1996.1489] [PMID:  8858125] 
[20] 
Zawarynski, P.; Tallerico, T.; Seeman, P.; Lee, S.P.; O’Dowd, B.F.; George, S.R. Dopamine D2 receptor dimers in human and rat brain. FEBS Lett.,  1998, 441(3), 383-386.
[http://dx.doi.org/10.1016/S0014-5793(98)01588-9] [PMID:  9891976] 
[21] 
Ciruela, F.; Casadó, V.; Mallol, J.; Canela, E.I.; Lluis, C.; Franco, R. Immunological identification of A1 adenosine receptors in brain cortex. J. Neurosci. Res.,  1995, 42(6), 818-828.
[http://dx.doi.org/10.1002/jnr.490420610] [PMID:  8847743] 
[22] 
Förster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Phys.,  1948, 2, 55-75.
[http://dx.doi.org/10.1002/andp.19484370105] 
[23] 
Ciruela, F.; Vilardaga, J.P.; Fernández-Dueñas, V. Lighting up multiprotein complexes: lessons from GPCR oligomerization. Trends Biotechnol.,  2010, 28(8), 407-415.
[http://dx.doi.org/10.1016/j.tibtech.2010.05.002] [PMID:  20542584] 
[24] 
Wu, B.; Chien, E.Y.T.; Mol, C.D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F.C.; Hamel, D.J.; Kuhn, P.; Handel, T.M.; Cherezov, V.; Stevens, R.C. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science,  2010, 330(6007), 1066-1071.
[http://dx.doi.org/10.1126/science.1194396] [PMID:  20929726] 
[25] 
Manglik, A.; Kruse, A.C.; Kobilka, T.S.; Thian, F.S.; Mathiesen, J.M.; Sunahara, R.K.; Pardo, L.; Weis, W.I.; Kobilka, B.K.; Granier, S. Crystal structure of the µ-opioid receptor bound to a morphinan antagonist. Nature,  2012, 485(7398), 321-326.
[http://dx.doi.org/10.1038/nature10954] [PMID:  22437502] 
[26] 
Huang, J.; Chen, S.; Zhang, J.J.; Huang, X.Y. Crystal structure of oligomeric β1-adrenergic G protein-coupled receptors in ligand-free basal state. Nat. Struct. Mol. Biol.,  2013, 20(4), 419-425.
[http://dx.doi.org/10.1038/nsmb.2504] [PMID:  23435379] 
[27] 
Ferré, S.; Fuxe, K.; von Euler, G.; Johansson, B.; Fredholm, B.B. Adenosine-dopamine interactions in the brain. Neuroscience,  1992, 51(3), 501-512.
[http://dx.doi.org/10.1016/0306-4522(92)90291-9] [PMID:  1488111] 
[28] 
Ferré, S.; von Euler, G.; Johansson, B.; Fredholm, B.B.; Fuxe, K. Stimulation of high-affinity adenosine A2 receptors decreases the affinity of dopamine D2 receptors in rat striatal membranes. Proc. Natl. Acad. Sci. USA,  1991, 88(16), 7238-7241.
[http://dx.doi.org/10.1073/pnas.88.16.7238] [PMID:  1678519] 
[29] 
Ferré, S.; Bonaventura, J.; Zhu, W.; Hatcher-Solis, C.; Taura, J.; Quiroz, C.; Cai, N-S.; Moreno, E.; Casadó-Anguera, V.; Kravitz, A.V.; Thompson, K.R.; Tomasi, D.G.; Navarro, G.; Cordomí, A.; Pardo, L.; Lluís, C.; Dessauer, C.W.; Volkow, N.D.; Casadó, V.; Ciruela, F.; Logothetis, D.E.; Zwilling, D. Essential control of the function of the striatopallidal neuron by pre-coupled complexes of adenosine A2A-Dopamine D2 receptor heterotetramers and adenylyl cyclase. Front. Pharmacol.,  2018, 9, 243.
[http://dx.doi.org/10.3389/fphar.2018.00243] [PMID:  29686613] 
[30] 
Fuxe, K.; Ferré, S.; Zoli, M.; Agnati, L.F. Integrated events in central dopamine transmission as analyzed at multiple levels. Evidence for intramembrane adenosine A2A/dopamine D2 and adenosine A1/dopamine D1 receptor interactions in the basal ganglia. Brain Res.,  1998, 26(2-3), 258-273.
[http://dx.doi.org/10.1016/S0165-0173(97)00049-0] [PMID:  9651540] 
[31] 
Canals, M.; Marcellino, D.; Fanelli, F.; Ciruela, F.; de Benedetti, P.; Goldberg, S.R.; Neve, K.; Fuxe, K.; Agnati, L.F.; Woods, A.S.; Ferré, S.; Lluis, C.; Bouvier, M.; Franco, R. Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer. J. Biol. Chem.,  2003, 278(47), 46741-46749.
[http://dx.doi.org/10.1074/jbc.M306451200] [PMID:  12933819] 
[32] 
Beggiato, S.; Antonelli, T.; Tomasini, M.C.; Borelli, A.C.; Agnati, L.F.; Tanganelli, S.; Fuxe, K.; Ferraro, L. Adenosine A2A-D2 receptor-receptor interactions in putative heteromers in the regulation of the striato-pallidal gaba pathway: possible relevance for parkinson’s disease and its treatment. Curr. Protein Pept. Sci.,  2014, 15(7), 673-680.
[http://dx.doi.org/10.2174/1389203715666140901103205] [PMID:  25175458] 
[33] 
Bonaventura, J.; Navarro, G.; Casadó-Anguera, V.; Azdad, K.; Rea, W.; Moreno, E.; Brugarolas, M.; Mallol, J.; Canela, E.I.; Lluís, C.; Cortés, A.; Volkow, N.D.; Schiffmann, S.N.; Ferré, S.; Casadó, V. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer. Proc. Natl. Acad. Sci. USA,  2015, 112(27), E3609-E3618.
[http://dx.doi.org/10.1073/pnas.1507704112] [PMID:  26100888] 
[34] 
Cabello, N.; Gandía, J.; Bertarelli, D.C.; Watanabe, M.; Lluís, C.; Franco, R.; Ferré, S.; Luján, R.; Ciruela, F. Metabotropic glutamate type 5, dopamine D2 and adenosine A2a receptors form higher-order oligomers in living cells. J. Neurochem.,  2009, 109(5), 1497-1507.
[http://dx.doi.org/10.1111/j.1471-4159.2009.06078.x] [PMID:  19344374] 
[35] 
Kamiya, T.; Saitoh, O.; Yoshioka, K.; Nakata, H. Oligomerization of adenosine A2A and dopamine D2 receptors in living cells. Biochem. Biophys. Res. Commun.,  2003, 306(2), 544-549.
[http://dx.doi.org/10.1016/S0006-291X(03)00991-4] [PMID:  12804599] 
[36] 
Ciruela, F.; Burgueño, J.; Casadó, V.; Canals, M.; Marcellino, D.; Goldberg, S.R.; Bader, M.; Fuxe, K.; Agnati, L.F.; Lluis, C.; Franco, R.; Ferré, S.; Woods, A.S. Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization. Direct epitope-epitope electrostatic interactions between adenosine A2A and dopamine D2 receptors. Anal. Chem.,  2004, 76(18), 5354-5363.
[http://dx.doi.org/10.1021/ac049295f] [PMID:  15362892] 
[37] 
Fernández-Dueñas, V.; Gómez-Soler, M.; Jacobson, K.A.; Kumar, S.T.; Fuxe, K.; Borroto-Escuela, D.O.; Ciruela, F. Molecular determinants of A2AR-D2R allosterism: role of the intracellular loop 3 of the D2R. J. Neurochem.,  2012, 123(3), 373-384.
[http://dx.doi.org/10.1111/j.1471-4159.2012.07956.x] [PMID:  22924752] 
[38] 
Borroto-Escuela, D.O.; Marcellino, D.; Narvaez, M.; Flajolet, M.; Heintz, N.; Agnati, L.; Ciruela, F.; Fuxe, K. A serine point mutation in the adenosine A2AR C-terminal tail reduces receptor heteromerization and allosteric modulation of the dopamine D2R. Biochem. Biophys. Res. Commun.,  2010, 394(1), 222-227.
[http://dx.doi.org/10.1016/j.bbrc.2010.02.168] [PMID:  20197060] 
[39] 
Morató, X.; Borroto-Escuela, D.O.; Fuxe, K.; Fernández-Dueñas, V.; Ciruela, F. Co-immunoprecipitation from brain. Neuromethods,  2016, 110, 19-30.
[http://dx.doi.org/10.1007/978-1-4939-3064-7_2] 
[40] 
Aguado, C.; Luján, R. The histoblot technique: a reliable approach to analyse expression profile of proteins and to predict their molecular association. Neuromethods,  2019, 144, 65-88.
[http://dx.doi.org/10.1007/978-1-4939-8985-0_6] 
[41] 
Aguado, C.; Orlandi, C.; Fajardo-Serrano, A.; Gil-Minguez, M.; Martemyanov, K.A.; Luján, R. Cellular and subcellular localization of the RGS7/Gβ5/R7BP complex in the cerebellar cortex. Front. Neuroanat.,  2016, 10, 114-130.
[http://dx.doi.org/10.3389/fnana.2016.00114] [PMID:  27965545] 
[42] 
Ciruela, F.; Fernández-Dueñas, V.; Sahlholm, K.; Fernández-Alacid, L.; Nicolau, J.C.; Watanabe, M.; Luján, R. Evidence for oligomerization between GABAB receptors and GIRK channels containing the GIRK1 and GIRK3 subunits. Eur. J. Neurosci.,  2010, 32(8), 1265-1277.
[http://dx.doi.org/10.1111/j.1460-9568.2010.07356.x] [PMID:  20846323] 
[43] 
Fernández-Alacid, L.; Watanabe, M.; Molnár, E.; Wickman, K.; Luján, R. Developmental regulation of G protein-gated inwardly-rectifying K+ (GIRK/Kir3) channel subunits in the brain. Eur. J. Neurosci.,  2011, 34(11), 1724-1736.
[http://dx.doi.org/10.1111/j.1460-9568.2011.07886.x] [PMID:  22098295] 
[44] 
Luján, R. International Review of Neurobiology; Academic Press Inc., 2015, Vol. 123, pp. 161-200.
[45] 
Bonaventura, J.; Rico, A.J.; Moreno, E.; Sierra, S.; Sánchez, M.; Luquin, N.; Farré, D.; Müller, C.E.; Martínez-Pinilla, E.; Cortés, A.; Mallol, J.; Armentero, M-T.; Pinna, A.; Canela, E.I.; Lluís, C.; McCormick, P.J.; Lanciego, J.L.; Casadó, V.; Franco, R. L-DOPA-treatment in primates disrupts the expression of A(2A) adenosine-CB(1) cannabinoid-D(2) dopamine receptor heteromers in the caudate nucleus. Neuropharmacology,  2014, 79, 90-100.
[http://dx.doi.org/10.1016/j.neuropharm.2013.10.036] [PMID:  24230991] 
[46] 
Zhu, Y.; Mészáros, J.; Walle, R.; Fan, R.; Sun, Z.; Dwork, A.J.; Trifilieff, P.; Javitch, J.A. Detecting G protein-coupled receptor complexes in postmortem human brain with proximity ligation assay and a Bayesian classifier. Biotechniques,  2020, 68(3), 122-129.
[http://dx.doi.org/10.2144/btn-2019-0083] [PMID:  31859535] 
[47] 
Fernández-Dueñas, V.; Gómez-Soler, M.; Valle-León, M.; Watanabe, M.; Ferrer, I.; Ciruela, F. Revealing adenosine A2A-dopamine D2 receptor heteromers in Parkinson’s disease post-mortem brain through a new alphascreen-based assay. Int. J. Mol. Sci.,  2019, 20(14), 3600-3611.
[http://dx.doi.org/10.3390/ijms20143600] [PMID:  31340557] 
[48] 
Luján, R.; Watanabe, M. Post-embedding immunohistochemistry in the localisation of receptors and ion channels. Neuromethods,  2016, 110, 211-232.
[http://dx.doi.org/10.1007/978-1-4939-3064-7_16] 
[49] 
Luján, R. Pre-embedding methods for the localization of receptors and ion channels. Neuromethods,  2016, 110, 191-210.
[http://dx.doi.org/10.1007/978-1-4939-3064-7_15] 
[50] 
Harada, H.; Shigemoto, R. High-resolution localization of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In: Neuromethods,  2016, 110, 233-245.
[51] 
Ries, J.; Kaplan, C.; Platonova, E.; Eghlidi, H.; Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods,  2012, 9(6), 582-584.
[http://dx.doi.org/10.1038/nmeth.1991] [PMID:  22543348] 
[52] 
Fernández-Dueñas, V.; Taura, J.J.; Cottet, M.; Gómez-Soler, M.; López-Cano, M.; Ledent, C.; Watanabe, M.; Trinquet, E.; Pin, J-P.; Luján, R.; Durroux, T.; Ciruela, F. Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats. Dis. Model. Mech.,  2015, 8(1), 57-63.
[http://dx.doi.org/10.1242/dmm.018143] [PMID:  25398851] 
[53] 
Söderberg, O.; Gullberg, M.; Jarvius, M.; Ridderstråle, K.; Leuchowius, K-J.; Jarvius, J.; Wester, K.; Hydbring, P.; Bahram, F.; Larsson, L-G.; Landegren, U. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods,  2006, 3(12), 995-1000.
[http://dx.doi.org/10.1038/nmeth947] [PMID:  17072308] 
[54] 
Trifilieff, P.; Rives, M-L.; Urizar, E.; Piskorowski, R.A.; Vishwasrao, H.D.; Castrillon, J.; Schmauss, C.; Slättman, M.; Gullberg, M.; Javitch, J.A. Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum. Biotechniques,  2011, 51(2), 111-118.
[http://dx.doi.org/10.2144/000113719] [PMID:  21806555] 
[55] 
Narváez, M.; Crespo-Ramírez, M.; Fores-Pons, R.; Pita-Rodríguez, M.; Ciruela, F.; Filip, M.; Beggiato, S.; Ferraro, L.; Tanganelli, S.; Ambrogini, P.; de la Mora, M.P.; Fuxe, K.; Borroto-Escuela, D.O. Study of GPCR homo- and heteroreceptor complexes in specific neuronal cell populations using the in situ proximity ligation assay. Neuromethods,  2021, 169, 117-134.
[http://dx.doi.org/10.1007/978-1-0716-1522-5_9] 
[56] 
Taura, J.; Fernández-Dueñas, V.; Ciruela, F. Visualizing G proteincoupled
receptor-receptor interactions in brain using proximity ligation
in situ assay. Curr. Protoc. Cell Biol,  2015, 67 17.1-17.17,
16.
[http://dx.doi.org/10.1002/0471143030.cb1717s67] [PMID:  26061241] 
[57] 
Borroto-Escuela, D.O.; Narvaez, M.; Valladolid-Acebes, I.; Shumilov, K.; Di Palma, M.; Wydra, K.; Schaefer, T.; Reyes-Resina, I.; Navarro, G.; Mudó, G.; Filip, M.; Sartini, S.; Friedland, K.; Schellekens, H.; Beggiato, S.; Ferraro, L.; Tanganelli, S.; Franco, R.; Belluardo, N.; Ambrogini, P.; de la Mora, M.; Fuxe, K. Detection, analysis, and quantification of GPCR homo- and heteroreceptor complexes in specific neuronal cell populations using the in situ proximity ligation assay. Neuromethods,  2018, 140, 299-315.
[http://dx.doi.org/10.1007/978-1-4939-8576-0_19] 
[58] 
López-Cano, M.; Fernández-Dueñas, V.; Ciruela, F. Proximity ligation assay image analysis protocol: Addressing receptor-receptor interactions. Methods Mol. Biol.,  2019, 2040, 41-50.
[http://dx.doi.org/10.1007/978-1-4939-9686-5_3] [PMID:  31432474] 
[59] 
Moreno-García, A.; Kun, A.; Calero, O.; Medina, M.; Calero, M. An overview of the role of lipofuscin in age-related neurodegeneration. Front. Neurosci.,  2018, 12, 464.
[http://dx.doi.org/10.3389/fnins.2018.00464] [PMID:  30026686] 
[60] 
Cardullo, R.A. Theoretical principles and practical considerations for fluorescence resonance energy transfer microscopy. Methods Cell Biol.,  2007, 81, 479-494.
[http://dx.doi.org/10.1016/S0091-679X(06)81023-X] [PMID:  17519181] 
[61] 
Stryer, L.; Haugland, R.P. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA,  1967, 58(2), 719-726.
[http://dx.doi.org/10.1073/pnas.58.2.719] [PMID:  5233469] 
[62] 
Stryer, L. Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem.,  1978, 47, 819-846.
[http://dx.doi.org/10.1146/annurev.bi.47.070178.004131] [PMID:  354506] 
[63] 
Gandía, J.; Lluís, C.; Ferré, S.; Franco, R.; Ciruela, F.; Gandia, J.; Lluis, C.; Ferre, S.; Franco, R.; Ciruela, F. Light resonance energy transfer-based methods in the study of G protein-coupled receptor oligomerization. BioEssays,  2008, 30(1), 82-89.
[http://dx.doi.org/10.1002/bies.20682] [PMID:  18081019] 
[64] 
Sheng, M.; Hoogenraad, C.C. The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu. Rev. Biochem.,  2007, 76, 823-847.
[http://dx.doi.org/10.1146/annurev.biochem.76.060805.160029] [PMID:  17243894] 
[65] 
Ferrandon, S.; Feinstein, T.N.; Castro, M.; Wang, B.; Bouley, R.; Potts, J.T.; Gardella, T.J.; Vilardaga, J.P. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat. Chem. Biol.,  2009, 5(10), 734-742.
[http://dx.doi.org/10.1038/nchembio.206] [PMID:  19701185] 
[66] 
Cottet, M.; Faklaris, O.; Maurel, D.; Scholler, P.; Doumazane, E.; Trinquet, E.; Pin, J.P.; Durroux, T. BRET and Time-resolved FRET strategy to study GPCR oligomerization: from cell lines toward native tissues. Front. Endocrinol. (Lausanne),  2012, 3, 92.
[67] 
Mathis, G. Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clin. Chem.,  1995, 41(9), 1391-1397.
[http://dx.doi.org/10.1093/clinchem/41.9.1391] [PMID:  7656455] 
[68] 
Bazin, H.; Trinquet, E.; Mathis, G. Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions. J. Biotechnol.,  2002, 82(3), 233-250.
[PMID:  11999692] 
[69] 
Terrillon, S.; Durroux, T.; Mouillac, B.; Breit, A.; Ayoub, M.A.; Taulan, M.; Jockers, R.; Barberis, C.; Bouvier, M. Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis. Mol. Endocrinol.,  2003, 17(4), 677-691.
[http://dx.doi.org/10.1210/me.2002-0222] [PMID:  12554793] 
[70] 
Navarro, G.; Cordomí, A.; Casadó-Anguera, V.; Moreno, E.; Cai, N-S.; Cortés, A.; Canela, E.I.; Dessauer, C.W.; Casadó, V.; Pardo, L.; Lluís, C.; Ferré, S. Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase. Nat. Commun.,  2018, 9(1), 1242.
[http://dx.doi.org/10.1038/s41467-018-03522-3] [PMID:  29593213] 
[71] 
Albizu, L.; Cottet, M.; Kralikova, M.; Stoev, S.; Seyer, R.; Brabet, I.; Roux, T.; Bazin, H.; Bourrier, E.; Lamarque, L.; Breton, C.; Rives, M.L.; Newman, A.; Javitch, J.; Trinquet, E.; Manning, M.; Pin, J.P.; Mouillac, B.; Durroux, T. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat. Chem. Biol.,  2010, 6(8), 587-594.
[http://dx.doi.org/10.1038/nchembio.396] [PMID:  20622858] 
[72] 
Hide, I.; Padgett, W.L.; Jacobson, K.A.; Daly, J.W.A. A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase. Mol. Pharmacol.,  1992, 41(2), 352-359.
[PMID:  1311411] 
[73] 
Fernández-Dueñas, V.; Durroux, T.; Ciruela, F. Receptor–Receptor Interactions in the Brain. Neuromethods,  2016, 110, 99-107.
[http://dx.doi.org/10.1007/978-1-4939-3064-7_8] 
[74] 
Valle-León, M.; Callado, L.F.; Aso, E.; Cajiao-Manrique, M.M.; Sahlholm, K.; López-Cano, M.; Soler, C.; Altafaj, X.; Watanabe, M.; Ferré, S.; Fernández-Dueñas, V.; Menchón, J.M.; Ciruela, F. Decreased striatal adenosine A2A-dopamine D2 receptor heteromerization in schizophrenia. Neuropsychopharmacology,  2021, 46(3), 665-672.
[http://dx.doi.org/10.1038/s41386-020-00872-9] [PMID:  33010795] 
[75] 
Kawano, K.; Yano, Y.; Omae, K.; Matsuzaki, S.; Matsuzaki, K. Stoichiometric analysis of oligomerization of membrane proteins on living cells using coiled-coil labeling and spectral imaging. Anal. Chem.,  2013, 85(6), 3454-3461.
[http://dx.doi.org/10.1021/ac400177a] [PMID:  23427815] 
[76] 
Felce, J.H.; Davis, S.J.; Klenerman, D. Single-molecule analysis of
G protein-coupled receptor stoichiometry: Approaches and limitations. Trends Pharmacol. Sci.,  2018, 39(2), 96-108.
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DOI https://dx.doi.org/10.2174/1570159X19666211104145727	Print ISSN 1570-159X
Publisher Name Bentham Science Publisher	Online ISSN 1875-6190
Current Neuropharmacology

Overcoming the Challenges of Detecting GPCR Oligomerization in the Brain

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