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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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.2014.11.112
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/10.1517/14728222.2014.981529
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):647
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.2018.04.002
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.2017.00309
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.7b00629
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, 2016
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.2015.244
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/B978-0-12-408143-7.00008-6
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.2012.05.007
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/B978-0-12-408143-7.00017-7
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/nmeth978
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 4
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Borroto-Escuela, D.O. et al. (2019). 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. In: Odagaki, Y., Borroto-Escuela, D. (eds) Co-Immunoprecipitation Methods for Brain Tissue . Neuromethods, vol 144. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8985-0_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8985-0_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8984-3
Online ISBN: 978-1-4939-8985-0
eBook Packages: Springer Protocols