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Co-immunoprecipitation of Membrane-Bound Receptors from Subsynaptic Compartments

  • Wilber Romero-FernandezEmail author
  • Maria Garcia-Barcelo
  • Yunis Perez-Betancourt
Protocol
Part of the Neuromethods book series (NM, volume 144)

Abstract

The allosteric modulation in G protein-coupled heteroreceptors have opened a new vision of the physiology and pathophysiology of the central nervous system. Understanding the role of the receptor-receptor interactions in the synaptic compartments is essential to elucidate the function of these macromolecular complexes in the brain intercellular communication that occurs within wiring and volume transmission. Here, we describe a procedure to identify the receptor-receptor interactions in different enriched subsynaptic fractions. First, we present a protocol to obtain synaptosomes by means of a discontinuous sucrose gradient. The method continues with a subcellular fractionation to separate the pre-synaptic, the post-synaptic, and the extra-synaptic fractions. Finally, we describe the Co-immunoprecipitation as an accepted technique to identify the receptor-receptor interaction from isolated and validated subsynaptic compartments.

Key words

G protein-coupled receptors Heteroreceptors Receptor-receptor interaction Synaptosome Sucrose gradient Subcellular fractionation Synaptic compartments Co-immunoprecipitation 

Notes

Acknowledgments

This work was supported by Prometeo Program of the Higher Education, Science, Technology and Innovation Secretary and the Technical University of Ambato (grants 1243-CU-P-2014 to WR-F, 0912-CU-P-2016 and 0932-CU-P-2016 to WR-F and YP-B, and 0906-CU-P-2016 to MG-B).

References

  1. 1.
    Fuxe K, Borroto-Escuela DO, Romero-Fernandez W, Palkovits M, Tarakanov AO, Ciruela F, Agnati LF (2014) Moonlighting proteins and protein-protein interactions as neurotherapeutic targets in the G protein-coupled receptor field. Neuropsychopharmacology 39(1):131–155.  https://doi.org/10.1038/npp.2013.242CrossRefPubMedGoogle Scholar
  2. 2.
    Fuxe K, Borroto-Escuela DO, Marcellino D, Romero-Fernandez W, Frankowska M, Guidolin D, Filip M, Ferraro L, Woods AS, Tarakanov A, Ciruela F, Agnati LF, Tanganelli S (2012) GPCR heteromers and their allosteric receptor-receptor interactions. Curr Med Chem 19(3):356–363CrossRefGoogle Scholar
  3. 3.
    Fuxe K, Borroto-Escuela DO, Romero-Fernandez W, Zhang WB, Agnati LF (2013) Volume transmission and its different forms in the central nervous system. Chin J Integr Med 19(5):323–329.  https://doi.org/10.1007/s11655-013-1455-1CrossRefPubMedGoogle Scholar
  4. 4.
    Phillips GR, Huang JK, Wang Y, Tanaka H, Shapiro L, Zhang W, Shan WS, Arndt K, Frank M, Gordon RE, Gawinowicz MA, Zhao Y, Colman DR (2001) The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution. Neuron 32(1):63–77CrossRefGoogle Scholar
  5. 5.
    Pinheiro PS, Rodrigues RJ, Silva AP, Cunha RA, Oliveira CR, Malva JO (2003) Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus. Neurosci Lett 336(2):97–100CrossRefGoogle Scholar
  6. 6.
    Klemmer P, Smit AB, Li KW (2009) Proteomics analysis of immuno-precipitated synaptic protein complexes. J Proteome 72(1):82–90.  https://doi.org/10.1016/j.jprot.2008.10.005CrossRefGoogle Scholar
  7. 7.
    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.  https://doi.org/10.1016/B978-0-12-391862-8.00015-6CrossRefPubMedGoogle Scholar
  8. 8.
    Canas PM, Cunha RA (2016) Subsynatic membrane fractionation. In: Receptor and ion channel detection in the brain, Methods and protocols, vol 110. Springer Science, New YorkCrossRefGoogle Scholar
  9. 9.
    Morató X, Borroto-Escuela DO, Fuxe K, Fernández-Dueñas V, Ciruela F (2016) Co-immunoprecipitation from brain, vol 110. Springer Science, New YorkGoogle Scholar
  10. 10.
    Gonder JC, Laber K (2007) A renewed look at laboratory rodent housing and management. ILAR J 48(1):29–36CrossRefGoogle Scholar
  11. 11.
    Romero-Fernandez W, Batista-Castro Z, De Lucca M, Ruano A, Garcia-Barcelo M, Rivera-Cervantes M, Garcia-Rodriguez J, Sanchez-Mateos S (2016) The 1, 2, 3 of laboratory animal experimentation. Revista peruana de medicina experimental y salud publica 33(2):288–299CrossRefGoogle Scholar
  12. 12.
    Li KW, Klemmer P, Smit AB (2010) Interaction proteomics of synapse protein complexes. Anal Bioanal Chem 397(8):3195–3202.  https://doi.org/10.1007/s00216-010-3658-zCrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Wilber Romero-Fernandez
    • 1
    • 2
    Email author
  • Maria Garcia-Barcelo
    • 2
  • Yunis Perez-Betancourt
    • 3
  1. 1.Department of Cell and Molecular BiologyUppsala UniversityUppsalaSweden
  2. 2.Faculty of Health SciencesTechnical University of AmbatoAmbatoEcuador
  3. 3.Faculty of Food Science and EngineeringTechnical University of AmbatoAmbatoEcuador

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