Measurement of agonist-stimulated [35S]GTPgammaS binding to assess total G-protein and Galpha-subtype-specific activation by G-protein-coupled receptors.

Abstract

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