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Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation

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A Correction to this article was published on 06 May 2021

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Abstract

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

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Abbreviations

CNS:

Central nervous system

GPCR:

G-protein-coupled receptors

GDP:

Guanosine diphosphate

GTP:

Guanosine triphosphate

NEM:

N-Ethylmaleimide

PTX:

Pertussis toxin

SPA:

Scintillation proximity assay

[35S]GTPγS:

Sulfur 35-labelled guanosine-5′-O-(γ-thio)-triphosphate

GTPγS:

Guanosine 5′-O-(3-triphosphate)

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Funding

This work was supported by the Spanish R + D + i Programme and the European Regional Development Funds (SAF2017-88126R), and the Basque Government (IT1211/19 to JJM; ELKARTEK Programme KK-2019/00049 to RD-A). IM-A was recipient of a fellowship from the Basque Government. RD-A, JJM and IM-A are members of the PSYBIAS Consortium, supported by Eranet Neuron Programme.

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Authors and Affiliations

  1. Department of Pharmacology, Centro de Investigación Biomédica en Red de Salud Mental, University of the Basque Country UPV/EHU CIBERSAM, 48940, Leioa, Bizkaia, Spain

    Rebeca Diez-Alarcia, Patricia Miranda-Azpiazu, Ane M. Gabilondo, J. Javier Meana & Itziar Muneta-Arrate

  2. Biocruces Bizkaia Health Research Institute, Barakaldo, Bizkaia, Spain

    Rebeca Diez-Alarcia, Ane M. Gabilondo & J. Javier Meana

  3. Department of Psychiatry, Faculty of Medicine, Saitama Medical University, 38 Morohongo Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan

    Yuji Odagaki

  4. Achucarro Basque Centre for Neuroscience, Leioa, Bizkaia, Spain

    Patricia Miranda-Azpiazu

Authors
  1. Rebeca Diez-Alarcia
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  2. Yuji Odagaki
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  3. Patricia Miranda-Azpiazu
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  4. Ane M. Gabilondo
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  5. J. Javier Meana
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  6. Itziar Muneta-Arrate
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Contributions

RD-A, YO and JMM contributed to the conception, design and supervision of the work. RD-A, YO, PM-A and IM-A undertook experimental procedures and data analyses included in the review. RD-A, AMG, JMM and IM-A drafted the first version of the manuscript. All authors contributed adding critical information in the different sections and helped to shape the manuscript. All authors approved the submitted version.

Corresponding author

Correspondence to Itziar Muneta-Arrate.

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Diez-Alarcia, R., Odagaki, Y., Miranda-Azpiazu, P. et al. Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation. Pharmacol. Rep 73, 1079–1095 (2021). https://doi.org/10.1007/s43440-021-00253-z

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  • DOI: https://doi.org/10.1007/s43440-021-00253-z

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