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[35S]GTPγS Binding as an Index of Total G-Protein and Gα-Subtype-Specific Activation by GPCRs

  • Rajendra Mistry
  • Mark R. Dowling
  • R. A. John ChallissEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 746)

Abstract

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through ­promoting guanine nucleotide exchange on Gα subunits of heterotrimeric G proteins to release Gα-GTP and free βγ-subunits. In membrane preparations, GTP can be substituted by 35S-labeled guanosine­ 5′-O-(3-thio)triphosphate ([35S]GTPγS) and on agonist stimulation a quasi-stable [35S]GTPγS–Gα ­complex forms and accumulates. Separation of [35S]GTPγS–Gα complexes from free [35S]GTPγS allows differences between basal and agonist-stimulated rates of [35S]GTPγS–Gα complex formation­ to be used to obtain pharmacological information on receptor–G-protein information transfer. Further, by releasing Gα-subunits into solution following the [35S]GTPγS binding step, Gα-subunit-specific antibodies can be used to investigate the Gα-protein subpopulations activated by receptors by immunoprecipitation of [35S]GTPγS–Gα complexes and quantification by scintillation counting. Here, we describe a total [35S]GTPγS binding assay and a modification of this method that incorporates a Gα-specific immunoprecipitation step.

Key words

[35S]GTPγS Guanine nucleotide binding protein G-protein-coupled receptor GDP Receptor–G-protein coupling Gα-protein-specific immunoprecipitation 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Rajendra Mistry
  • Mark R. Dowling
  • R. A. John Challiss
    • 1
    Email author
  1. 1.Department of Cell Physiology and PharmacologyUniversity of LeicesterLeicesterUK

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