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Study of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity Ligation Assay

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Receptor and Ion Channel Detection in the Brain

Part of the book series: Neuromethods ((NM,volume 169))

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.

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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.

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Authors and Affiliations

  1. Instituto de Investigación Biomédica de Málaga, Facultad de Medicina, Universidad de Málaga, Málaga, Spain

    Manuel Narváez, Ramon Fores-Pons & Mariana Pita-Rodríguez

  2. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

    Minerva Crespo-Ramírez & Miguel Perez de la Mora

  3. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    Ramon Fores-Pons, Mariana Pita-Rodríguez, Kjell Fuxe & Dasiel O. Borroto-Escuela

  4. Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, Universitat de Barcelona, IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain

    Francisco Ciruela

  5. Institut de Neurociències, Universitat de Barcelona, Barcelona, Spain

    Francisco Ciruela

  6. Laboratory of Drug Addiction Pharmacology, Polish Academy of Sciences, Institute of Pharmacology, Kraków, Poland

    Malgorzata Filip

  7. Department of Medical Sciences, University of Ferrara, Ferrara, Italy

    Sarah Beggiato & Luca Ferraro

  8. Department of Life Sciences and Biotechnology (SVEB), University of Ferrara, Ferrara, Italy

    Sergio Tanganelli

  9. Section of Physiology, Department of Biomolecular Science, University of Urbino, Urbino, Italy

    Patrizia Ambrogini & Dasiel O. Borroto-Escuela

  10. Observatorio Cubano de Neurociencias, Grupo Bohío-Estudio, Yaguajay, Cuba

    Dasiel O. Borroto-Escuela

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  1. Manuel Narváez
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  2. Minerva Crespo-Ramírez
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  11. Miguel Perez de la Mora
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  12. Kjell Fuxe
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  13. Dasiel O. Borroto-Escuela
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Corresponding author

Correspondence to Dasiel O. Borroto-Escuela .

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Editors and Affiliations

  1. Instituto de Investigación en Discapacidades Neurológicas (IDINE), Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain

    Rafael Lujan

  2. Pharmacology Unit, Department of Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, IDIBELL, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain

    Francisco Ciruela

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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

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  • DOI: https://doi.org/10.1007/978-1-0716-1522-5_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1521-8

  • Online ISBN: 978-1-0716-1522-5

  • eBook Packages: Springer Protocols

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