title: 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.
authors.affiliation: Novartis Horsham Research Center, West Sussex, United Kingdom.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Dowling
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Nahorski
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000819:agonists / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000378:metabolism*; D013462:Sulfur Radioisotopes
chemical_list: D043562:Receptors, G-Protein-Coupled; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins