Abstract
Membrane receptor, for example, G-protein-coupled receptors (GPCRs), operates via coordinated changes between the receptor expression, their modifications, and interactions between each other. Perturbation in specific heteroreceptor complexes and/or their balance/equilibrium with other heteroreceptor complexes and corresponding homoreceptor complexes is considered to have a role in pathogenic mechanisms, including drug addiction, depression, Parkinson’s disease, and schizophrenia. To understand the associations of GPCRs and to unravel the global picture of their receptor–receptor interactions in the brain, different experimental detection techniques for receptor–receptor interactions have been established (e.g., co-immunoprecipitation-based approach). However, they have been criticized for not reflecting the cellular situation or the dynamic nature of receptor–receptor interactions. Therefore, the detection and visualization of GPCR homo- and heteroreceptor complexes in the brain remained largely unknown until recent years, when a well-characterized in situ proximity ligation assay (in situ PLA) was adapted to validate the receptor complexes in their native environment. The in situ PLA protocol presented here can be used to visualize GPCR receptor–receptor interactions in cells and tissues in a highly sensitive and specific manner. We have developed a combined method using immunohistochemistry and PLA, particularly aimed to monitor interactions between GPCRs in specific neuronal cell populations. This allows the analysis of homo- and heteroreceptor complexes at a cellular and subcellular level. The method has the advantage that it can be used in clinical specimens, providing localized, quantifiable homo and heteroreceptor complexes detected in single cells. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research. We discuss also their potential as tools for drug development and diagnostics.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Borroto-Escuela DO, Wydra K, Pintsuk J, Narvaez M, Corrales F, Zaniewska M, Agnati LF, Franco R, Tanganelli S, Ferraro L et al (2016) Understanding the functional plasticity in neural networks of the basal ganglia in cocaine use disorder: a role for allosteric receptor-receptor interactions in a2a-d2 heteroreceptor complexes. Neural Plast 2016:4827268
Borroto-Escuela DO, Wydra K, Ferraro L, Rivera A, Filip M, Fuxe K (2015) Role of d2-like heteroreceptor compelxes in the effects of cocaine, morphine and hallucinogens. In: Preedy V (ed) Neurophatology of drug addictions and substance misuse, vol 1. Elsevier, London, pp 93–101
Bjork K, Svenningsson P (2011) Modulation of monoamine receptors by adaptor proteins and lipid rafts: role in some effects of centrally acting drugs and therapeutic agents. Annu Rev Pharmacol Toxicol 51:211–242
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
Aarsland D, Pahlhagen S, Ballard CG, Ehrt U, Svenningsson P (2011) Depression in parkinson disease--epidemiology, mechanisms and management. Nat Rev Neurol 8:35–47
Artigas F (2015) Developments in the field of antidepressants, where do we go now? Eur Neuropsychopharmacol 25:657–670
Blier P (2013) Neurotransmitter targeting in the treatment of depression. J Clin Psychiatry 74(Suppl 2):19–24
Borroto-Escuela DO, Fuxe K (2017) Diversity and bias through dopamine d2r heteroreceptor complexes. Curr Opin Pharmacol 32:16–22
Fuxe K, Borroto-Escuela DO (2016) Heteroreceptor complexes and their allosteric receptor-receptor interactions as a novel biological principle for integration of communication in the cns: targets for drug development. Neuropsychopharmacology 41:380–382
Borroto-Escuela DO, Brito I, Di Palma M, Jiménez-Beristain A, Narvaez M, Corrales F, Pita-Rodríguez M, Sartini S, Ambrogini P, Lattanzi D et al (2015) On the role of the balance of gpcr homo/ heteroreceptor complexes in the brain. J Adv Neurosci Res 2:36–44
Borroto-Escuela DO, Narvaez M, Perez-Alea M, Tarakanov AO, Jimenez-Beristain A, Mudo G, Agnati LF, Ciruela F, Belluardo N, Fuxe K (2015) Evidence for the existence of fgfr1-5-ht1a heteroreceptor complexes in the midbrain raphe 5-ht system. Biochem Biophys Res Commun 456:489–493
Fuxe K, Guidolin D, Agnati LF, Borroto-Escuela DO (2015) Dopamine heteroreceptor complexes as therapeutic targets in parkinson’s disease. Expert Opin Ther Targets 19:377–398
Fuxe K, Agnati LF, Borroto-Escuela DO (2014) The impact of receptor-receptor interactions in heteroreceptor complexes on brain plasticity. Expert Rev Neurother 14:719–721
Fuxe K, Borroto-Escuela D, Fisone G, Agnati LF, Tanganelli S (2014) Understanding the role of heteroreceptor complexes in the central nervous system. Curr Protein Pept Sci 15:647
Fuxe K, Borroto-Escuela DO, Ciruela F, Guidolin D, Agnati LF (2014) Receptor-receptor interactions in heteroreceptor complexes: A new principle in biology. Focus on their role in learning and memory. Neurosci Discov:2
Fuxe K, Tarakanov A, Romero Fernandez W, Ferraro L, Tanganelli S, Filip M, Agnati LF, Garriga P, Diaz-Cabiale Z, Borroto-Escuela DO (2014) Diversity and bias through receptor-receptor interactions in gpcr heteroreceptor complexes. Focus on examples from dopamine d2 receptor heteromerization. Front Endocrinol 5:71
Borroto-Escuela DO, Narvaez M, Perez-Alea M, Tarakanov AO, Jimenez-Beristain A, Mudo G, Agnati LF, Ciruela F, Belluardo N, Fuxe K (2014) Evidence for the existence of fgfr1-5-ht1a heteroreceptor complexes in the midbrain raphe 5-ht system. Biochem Biophys Res Commun
Borroto-Escuela DO, Narvaez M, Wydra K, Pintsuk J, Pinton L, Jimenez-Beristain A, Di Palma M, Jastrzebska J, Filip M, Fuxe K (2017) Cocaine self-administration specifically increases a2ar-d2r and d2r-sigma1r heteroreceptor complexes in the rat nucleus accumbens shell. Relevance for cocaine use disorder. Pharmacol Biochem Behav 155:24–31
Pintsuk J, Borroto-Escuela DO, Lai TK, Liu F, Fuxe K (2016) Alterations in ventral and dorsal striatal allosteric a2ar-d2r receptor-receptor interactions after amphetamine challenge: relevance for schizophrenia. Life Sci 167:92–97
Pintsuk J, Borroto-Escuela DO, Pomierny B, Wydra K, Zaniewska M, Filip M, Fuxe K (2016) Cocaine self-administration differentially affects allosteric a2a-d2 receptor-receptor interactions in the striatum. Relevance for cocaine use disorder. Pharmacol Biochem Behav 144:85–91
Narvaez M, Millon C, Borroto-Escuela D, Flores-Burgess A, Santin L, Parrado C, Gago B, Puigcerver A, Fuxe K, Narvaez JA et al (2014) Galanin receptor 2-neuropeptide y y1 receptor interactions in the amygdala lead to increased anxiolytic actions. Brain Struct Funct
Borroto-Escuela DO, Romero-Fernandez W, Narvaez M, Oflijan J, Agnati LF, Fuxe K (2014) Hallucinogenic 5-ht2ar agonists lsd and doi enhance dopamine d2r protomer recognition and signaling of d2-5-ht2a heteroreceptor complexes. Biochem Biophys Res Commun 443:278–284
Borroto-Escuela DO, Narvaez M, Di Palma M, Calvo F, Rodriguez D, Millon C, Carlsson J, Agnati LF, Garriga P, Diaz-Cabiale Z et al (2014) Preferential activation by galanin 1-15 fragment of the galr1 protomer of a galr1-galr2 heteroreceptor complex. Biochem Biophys Res Commun 452:347–353
Millon C, Flores-Burgess A, Narvaez M, Borroto-Escuela DO, Santin L, Parrado C, Narvaez JA, Fuxe K, Diaz-Cabiale Z (2014) A role for galanin n-terminal fragment (1-15) in anxiety- and depression-related behaviours in rats. Int J Neuropsychopharmacol
Borroto-Escuela DO, Romero-Fernandez W, Mudo G, Perez-Alea M, Ciruela F, Tarakanov AO, Narvaez M, Di Liberto V, Agnati LF, Belluardo N et al (2012) Fibroblast growth factor receptor 1–5-hydroxytryptamine 1a heteroreceptor complexes and their enhancement of hippocampal plasticity. Biol Psychiatry 71:84–91
Flajolet M, Wang Z, Futter M, Shen W, Nuangchamnong N, Bendor J, Wallach I, Nairn AC, Surmeier DJ, Greengard P (2008) Fgf acts as a co-transmitter through adenosine a(2a) receptor to regulate synaptic plasticity. Nat Neurosci 11:1402–1409
Nai Q, Li S, Wang SH, Liu J, Lee FJ, Frankland PW, Liu F (2009) Uncoupling the d1-n-methyl-d-aspartate (nmda) receptor complex promotes nmda-dependent long-term potentiation and working memory. Biol Psychiatry 67:246–254
Borroto-Escuela DO, Romero-Fernandez W, Tarakanov AO, Ciruela F, Agnati LF, Fuxe K (2011) On the existence of a possible a2a-d2-beta-arrestin2 complex: A2a agonist modulation of d2 agonist-induced beta-arrestin2 recruitment. J Mol Biol 406:687–699
Laroche G, Lepine MC, Theriault C, Giguere P, Giguere V, Gallant MA, de Brum-Fernandes A, Parent JL (2005) Oligomerization of the alpha and beta isoforms of the thromboxane a2 receptor: relevance to receptor signaling and endocytosis. Cell Signal 17:1373–1383
Borroto-Escuela DO, Garcia-Negredo G, Garriga P, Fuxe K, Ciruela F (1803) The m(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. Biochim Biophys Acta 2010:813–825
Borroto-Escuela DO, Van Craenenbroeck K, Romero-Fernandez W, Guidolin D, Woods AS, Rivera A, Haegeman G, Agnati LF, Tarakanov AO, Fuxe K (2010) Dopamine d2 and d4 receptor heteromerization and its allosteric receptor-receptor interactions. Biochem Biophys Res Commun 404:928–934
Comps-Agrar L, Maurel D, Rondard P, Pin JP, Trinquet E, Prezeau L (2011) Cell-surface protein-protein interaction analysis with time-resolved fret and snap-tag technologies: application to g protein-coupled receptor oligomerization. Methods Mol Biol 756:201–214
Cottet M, Faklaris O, Falco A, Trinquet E, Pin JP, Mouillac B, Durroux T (2013) Fluorescent ligands to investigate gpcr binding properties and oligomerization. Biochem Soc Trans 41:148–153
Cottet M, Faklaris O, Maurel D, Scholler P, Doumazane E, Trinquet E, Pin JP, Durroux T (2012) Bret and time-resolved fret strategy to study gpcr oligomerization: from cell lines toward native tissues. Front Endocrinol 3:92
Schellekens H, De Francesco PN, Kandil D, Theeuwes WF, McCarthy T, van Oeffelen WE, Perello M, Giblin L, Dinan TG, Cryan JF (2015) Ghrelin’s orexigenic effect is modulated via a serotonin 2c receptor interaction. ACS Chem Neurosci 6:1186–1197
Borroto-Escuela DO, Flajolet M, Agnati LF, Greengard P, Fuxe K (2013) Bioluminescence resonance energy transfer methods to study g protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes. Methods Cell Biol 117:141–164
Borroto-Escuela DO, Romero-Fernandez W, Tarakanov AO, Marcellino D, Ciruela F, Agnati LF, Fuxe K (2010) Dopamine d2 and 5-hydroxytryptamine 5-ht((2)a) receptors assemble into functionally interacting heteromers. Biochem Biophys Res Commun 401:605–610
Bouvier M, Heveker N, Jockers R, Marullo S, Milligan G (2007) Bret analysis of gpcr oligomerization: newer does not mean better. Nat Methods 4:3–4. author reply 4
James JR, Oliveira MI, Carmo AM, Iaboni A, Davis SJ (2006) A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat Methods 3:1001–1006
Marullo S, Bouvier M (2007) Resonance energy transfer approaches in molecular pharmacology and beyond. Trends Pharmacol Sci 28:362–365
Audet M, Lagace M, Silversides DW, Bouvier M (2010) Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer. FASEB J 24:2829–2838
Fernandez-Duenas V, Gomez-Soler M, Jacobson KA, Santhosh Kumar T, Fuxe K, Borroto-Escuela DO, Ciruela F (2012) Molecular determinants of a(2a) r-d(2) r allosterism: role of the intracellular loop 3 of the d(2) r. J Neurochem
Herrick-Davis K, Grinde E, Cowan A, Mazurkiewicz JE (2013) Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle. Mol Pharmacol 84:630–642
Borroto-Escuela DO, Romero-Fernandez W, Garriga P, Ciruela F, Narvaez M, Tarakanov AO, Palkovits M, Agnati LF, Fuxe K (2013) G protein-coupled receptor heterodimerization in the brain. Methods Enzymol 521:281–294
Trifilieff P, Rives ML, Urizar E, Piskorowski RA, Vishwasrao HD, Castrillon J, Schmauss C, Slattman M, Gullberg M, Javitch JA (2011) Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine d2-adenosine a2a receptor complexes in the striatum. Biotechniques 51:111–118
Romero-Fernandez W, Borroto-Escuela DO, Agnati LF, Fuxe K (2013) Evidence for the existence of dopamine d2-oxytocin receptor heteromers in the ventral and dorsal striatum with facilitatory receptor-receptor interactions. Mol Psychiatry 18:849–850
Borroto-Escuela DO, Li X, Tarakanov AO, Savelli D, Narvaez M, Shumilov K, Andrade-Talavera Y, Jimenez-Beristain A, Pomierny B, Diaz-Cabiale Z et al (2017) Existence of brain 5-ht1a-5-ht2a isoreceptor complexes with antagonistic allosteric receptor-receptor interactions regulating 5-ht1a receptor recognition. ACS Omega 2:4779–4789
Borroto-Escuela DO, DuPont CM, Li X, Savelli D, Lattanzi D, Srivastava I, Narvaez M, Di Palma M, Barbieri E, Andrade-Talavera Y et al (2017) Disturbances in the fgfr1-5-ht1a heteroreceptor complexes in the raphe-hippocampal 5-ht system develop in a genetic rat model of depression. Front Cell Neurosci 11:309
Fuxe K, Borroto-Escuela DO (2018) Receptor-receptor interactions in the central nervous system, vol 140. Humana Press, New York, p 346
Borroto-Escuela DO, Tarakanov AO, Fuxe K (2016) Fgfr1-5-ht1a heteroreceptor complexes: implications for understanding and treating major depression. Trends Neurosci 39:5–15
Borroto-Escuela DO, Hagman B, Woolfenden M, Pinton L, Jiménez-Beristain A, Oflijan J, Narvaez M, Di Palma M, Feltmann K, Sartini S et al (2016) In situ proximity ligation assay to study and understand the distribution and balance of gpcr homo- and heteroreceptor complexes in the brain. In: Lujan R, Ciruela F (eds) Receptor and ion channel detection in the brain, vol 110. Springer, Berlin, pp 109–126
Borroto-Escuela DO, Wydra K, Filip M, Fuxe K (2018) A2ar-d2r heteroreceptor complexes in cocaine reward and addiction. Trends Pharmacol Sci 39:1008–1020
Borroto-Escuela DO, Wydra K, Li X, Rodriguez D, Carlsson J, Jastrzebska J, Filip M, Fuxe K (2018) Disruption of a2ar-d2r heteroreceptor complexes after a2ar transmembrane 5 peptide administration enhances cocaine self-administration in rats. Mol Neurobiol 55:7038–7048
Millon C, Flores-Burgess A, Narvaez M, Borroto-Escuela DO, Santin L, Gago B, Narvaez JA, Fuxe K, Diaz-Cabiale Z (2016) Galanin (1–15) enhances the antidepressant effects of the 5-ht1a receptor agonist 8-oh-dpat. Involvement of the raphe-hippocampal 5-ht neuron system. Brain Struct Funct 221(9):4491–4504
Tena-Campos M, Ramon E, Borroto-Escuela DO, Fuxe K, Garriga P (1852) The zinc binding receptor gpr39 interacts with 5-ht1a and galr1 to form dynamic heteroreceptor complexes with signaling diversity. Biochim Biophys Acta 2015:2585–2592
Pinton L, Borroto-Escuela DO, Narváez M, Jiménez-Beristain A, Oflijan J, Ferraro L, Agnati LF, Fuxe K (2015) Dopamine d2 receptor dynamic and modulation in the d2r-sigma1r heteroreceptor complexes: role in cocaine actions. In: European neuropsychopharmacology, vol 25. Elsevier, Amsterdam, The Netherlands, pp S609–S610
Borroto-Escuela DO, Perez De La Mora M, Manger P, Narvaez M, Beggiato S, Crespo-Ramirez M, Navarro G, Wydra K, Diaz-Cabiale Z, Rivera A et al (2018) Brain dopamine transmission in health and parkinson’s disease: Modulation of synaptic transmission and plasticity through volume transmission and dopamine heteroreceptors. Front Syn Neurosci 10:20
Borroto-Escuela DO, Carlsson J, Ambrogini P, Narvaez M, Wydra K, Tarakanov AO, Li X, Millon C, Ferraro L, Cuppini R et al (2017) Understanding the role of gpcr heteroreceptor complexes in modulating the brain networks in health and disease. Front Cell Neurosci 11:37
Borroto-Escuela DO, Pintsuk J, Schafer T, Friedland K, Ferraro L, Tanganelli S, Liu F, Fuxe K (2016) Multiple d2 heteroreceptor complexes: new targets for treatment of schizophrenia. Ther Adva Psychopharmacol 6:77–94
Navarro G, Borroto-Escuela DO, Fuxe K, Franco R (2016) Purinergic signaling in parkinson’s disease. v. Neuropharmacology 104:161–168
Ferraro L, Beggiato S, Borroto-Escuela DO, Ravani L, O’Connor WT, Tomasini MC, Borelli AC, Agnati LF, Antonelli T, Tanganelli S et al (2014) Neurotensin nts1-dopamine d2 receptor-receptor interactions in putative receptor heteromers: relevance for parkinson’s disease and schizophrenia. Curr Protein Pept Sci 15:681–690
Borroto-Escuela DO, Agnati LF, Fuxe K, Ciruela F (2012) Muscarinic acetylcholine receptor-interacting proteins (machrips): targeting the receptorsome. Curr Drug Targets 13:53–71
Borroto-Escuela DO, Correia PA, Romero-Fernandez W, Narvaez M, Fuxe K, Ciruela F, Garriga P (2011) Muscarinic receptor family interacting proteins: role in receptor function. J Neurosci Methods 195:161–169
Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20:473–477
Gullberg M, Fredriksson S, Taussig M, Jarvius J, Gustafsdottir S, Landegren U (2003) A sense of closeness: protein detection by proximity ligation. Curr Opin Biotechnol 14:82–86
Gullberg M, Gustafsdottir SM, Schallmeiner E, Jarvius J, Bjarnegard M, Betsholtz C, Landegren U, Fredriksson S (2004) Cytokine detection by antibody-based proximity ligation. Proc Natl Acad Sci U S A 101:8420–8424
Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000
Darmanis S, Kahler A, Spangberg L, Kamali-Moghaddam M, Landegren U, Schallmeiner E (2007) Self-assembly of proximity probes for flexible and modular proximity ligation assays. Biotechniques 43:443–444. 446, 448 passim
Soderberg O, Leuchowius KJ, Kamali-Moghaddam M, Jarvius M, Gustafsdottir S, Schallmeiner E, Gullberg M, Jarvius J, Landegren U (2007) Proximity ligation: a specific and versatile tool for the proteomic era. Genet Eng (N Y) 28:85–93
Soderberg O, Leuchowius KJ, Gullberg M, Jarvius M, Weibrecht I, Larsson LG, Landegren U (2008) Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45:227–232
Uhlen M, Bandrowski A, Carr S, Edwards A, Ellenberg J, Lundberg E, Rimm DL, Rodriguez H, Hiltke T, Snyder M et al (2016) A proposal for validation of antibodies. Nat Methods 13:823–827
Antonelli T, Fuxe K, Agnati L, Mazzoni E, Tanganelli S, Tomasini MC, Ferraro L (2006) Experimental studies and theoretical aspects on a2a/d2 receptor interactions in a model of parkinson’s disease. Relevance for l-dopa induced dyskinesias. J Neurol Sci 248:16–22
Weibrecht I, Leuchowius KJ, Clausson CM, Conze T, Jarvius M, Howell WM, Kamali-Moghaddam M, Soderberg O (2010) Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Rev Proteomics 7:401–409
Acknowledgments
This work has been supported by the Karolinska Institutets Forskningsstiftelser 2020-2021 and Hjärnfonden (FO-2018-286 and FO2019-296) to D.O.B.-E., by the Swedish Medical Research Council (62X-00715-50-3) and Stiftelsen Olle Engkvist Byggmästare to K.F. K.F. and D.O.B.-E., and by Dirección General de Apoyo al Personal Académico (DGAPA), Universidad Nacional Autónoma de México (IN206820) to M.P.M. D.O.B.-E. belongs to Academia de Biólogos Cubanos.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Narváez, M. et al. (2021). Study of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity Ligation Assay. In: Lujan, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1522-5_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1522-5_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1521-8
Online ISBN: 978-1-0716-1522-5
eBook Packages: Springer Protocols