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Isolation and Detection of G Protein-Coupled Receptor (GPCR) Heteroreceptor Complexes in Rat Brain Synaptosomal Preparation Using a Combined Brain Subcellular Fractionation/Co-immunoprecipitation (Co-IP) Procedures

  • Dasiel O. Borroto-Escuela
  • Manuel Narvaez
  • Martina Zannoni
  • Chiara Contri
  • Minerva Crespo-Ramírez
  • Michael di Palma
  • Patrizia Ambrogini
  • Daily Y. Borroto-Escuela
  • Ismel Brito
  • Mariana Pita-Rodríguez
  • Ismael Valladolid-Acebes
  • Miguel Pérez de la Mora
  • Kjell FuxeEmail author
Protocol
Part of the Neuromethods book series (NM, volume 144)

Abstract

The isolation and characterization of GPCR heteroreceptor complexes, specially those present at the central nervous system, are of crucial relevance for the understanding of the molecular mechanisms behind several mental and neurodegenerative disorders. The existence of homo- and heteroreceptor complexes with allosteric receptor-receptor interactions increases the diversity of receptor function including recognition, trafficking, and signaling. This phenomenon increases our understanding of how brain function is altered through molecular integration of receptor signals. An alteration in specific heteroreceptor complexes or their neuronal localization is considered to have a role in the pathogenic mechanisms that lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. Therefore, it is fundamental to understand the appropriate localization and synaptic clustering of these GPCR heteroreceptor complexes. This chapter represents a workflow for the analysis of GPCR heteroreceptor complexes by means of combined use of differential centrifugation/co-immunoprecipitation in rat brain tissue. The combination of differential centrifugation/co-immunoprecipitation allows the separation and detection of GPCR heteroreceptor complexes present at synaptic sites from those found in intracellular compartments and vesicular pools. It is a reproducible protocol and produces reliable quantitative data.

Key words

G protein-coupled receptors (GPCRs) GPCR heteroreceptor complexes Receptor-receptor interactions Heterodimerization Co-immunoprecipitation Subcellular Synaptic membrane protein Ultracentrifugation 

Notes

Acknowledgments

This work has been supported by the Karolinska Institutets Forskningsstiftelser 2014/2015 to DOBE, by the Swedish Medical Research Council (62X-00715-50-3) and AFA Försäkring (130328) to KF and DOBE. DOBE belongs to Academia de Biólogos Cubanos.

References

  1. 1.
    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 456(1):489–493.  https://doi.org/10.1016/j.bbrc.2014.11.112CrossRefPubMedGoogle Scholar
  2. 2.
    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(3):377–398.  https://doi.org/10.1517/14728222.2014.981529CrossRefPubMedGoogle Scholar
  3. 3.
    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(7):647CrossRefGoogle Scholar
  4. 4.
    Borroto-Escuela DO, Tarakanov AO, Brito I, Fuxe K (2018) Glutamate heteroreceptor complexes in the brain. Pharmacol Rep 70(5):936–950.  https://doi.org/10.1016/j.pharep.2018.04.002CrossRefGoogle Scholar
  5. 5.
    Borroto-Escuela DO, DuPont CM, Li X, Savelli D, Lattanzi D, Srivastava I, Narvaez M, Di Palma M, Barbieri E, Andrade-Talavera Y, Cuppini R, Odagaki Y, Palkovits M, Ambrogini P, Lindskog M, Fuxe K (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.  https://doi.org/10.3389/fncel.2017.00309CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Borroto-Escuela DO, Li X, Tarakanov AO, Savelli D, Narvaez M, Shumilov K, Andrade-Talavera Y, Jimenez-Beristain A, Pomierny B, Diaz-Cabiale Z, Cuppini R, Ambrogini P, Lindskog M, Fuxe K (2017) Existence of brain 5-HT1A-5-HT2A isoreceptor complexes with antagonistic allosteric receptor-receptor interactions regulating 5-HT1A receptor recognition. ACS Omega 2(8):4779–4789.  https://doi.org/10.1021/acsomega.7b00629CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Borroto-Escuela DO, Narváez M, Jiménez-Beristain A, Fuxe K (2016) FGFR1–5-HT1A heteroreceptor complexes in the hippocampus and midbrain raphe as a novel targets for antidepressant drugs. Paper presented at the 30th CINP World Congress of Neuropsycho-pharmacology, Seoul, Republic of Korea, July 5, 2016Google Scholar
  8. 8.
    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(1):380–382.  https://doi.org/10.1038/npp.2015.244CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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.  https://doi.org/10.1016/B978-0-12-408143-7.00008-6CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Fernandez-Duenas V, Llorente J, Gandia J, Borroto-Escuela DO, Agnati LF, Tasca CI, Fuxe K, Ciruela F (2012) Fluorescence resonance energy transfer-based technologies in the study of protein-protein interactions at the cell surface. Methods 57(4):467–472.  https://doi.org/10.1016/j.ymeth.2012.05.007CrossRefPubMedGoogle Scholar
  11. 11.
    Skieterska K, Duchou J, Lintermans B, Van Craenenbroeck K (2013) Detection of G protein-coupled receptor (GPCR) dimerization by coimmunoprecipitation. Methods Cell Biol 117:323–340.  https://doi.org/10.1016/B978-0-12-408143-7.00017-7CrossRefPubMedGoogle Scholar
  12. 12.
    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(12):1001–1006.  https://doi.org/10.1038/nmeth978CrossRefPubMedGoogle Scholar
  13. 13.
    Bouvier M, Heveker N, Jockers R, Marullo S, Milligan G (2007) BRET analysis of GPCR oligomerization: newer does not mean better. Nat Methods 4(1):3–4.  https://doi.org/10.1038/nmeth0107-3; author reply 4CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Dasiel O. Borroto-Escuela
    • 1
    • 2
  • Manuel Narvaez
    • 3
  • Martina Zannoni
    • 1
    • 4
  • Chiara Contri
    • 1
    • 4
  • Minerva Crespo-Ramírez
    • 5
  • Michael di Palma
    • 6
  • Patrizia Ambrogini
    • 6
  • Daily Y. Borroto-Escuela
    • 2
    • 7
  • Ismel Brito
    • 1
    • 2
  • Mariana Pita-Rodríguez
    • 3
    • 8
  • Ismael Valladolid-Acebes
    • 9
  • Miguel Pérez de la Mora
    • 5
  • Kjell Fuxe
    • 1
    Email author
  1. 1.Department of NeuroscienceKarolinska InstitutetStockholmSweden
  2. 2.Observatorio Cubano de NeurocienciasYaguajayCuba
  3. 3.Facultad de Medicina, Instituto de Investigación Biomédica de Málaga, Universidad de MálagaMálagaSpain
  4. 4.Department of Life Science and BiotechnologyUniversity of FerraraFerraraItaly
  5. 5.Instituto de Fisiología CelularUniversidad Nacional Autónoma de MéxicoMexico CityMexico
  6. 6.Section of Physiology, Department of Biomolecular ScienceUniversity of UrbinoUrbinoItaly
  7. 7.Environmental Services Center, Caguanes National ParkMinistry of Science, Technology and Environment (CITMA)YaguajayCuba
  8. 8.Neurogenetics DepartmentInstitute of Neurology and NeurosurgeryHavanaCuba
  9. 9.The Rolf Luft Research Center for Diabetes and EndocrinologyKarolinska Institutet, Karolinska University Hospital L1StockholmSweden

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