Analysis of receptor-stimulated and basal guanine nucleotide binding to membrane G proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A method to study [alpha-32P]GTP binding to the alpha subunit of GTP-binding proteins in rat brain membranes is described. This method measures receptor-stimulated GTP binding to individual alpha subunits. GTP binding is associated with two protein bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The bands, 40- and 45-kDa in size, comigrate with the alpha subunits of Gi/Go and Gs, respectively. Binding of [alpha-32P]GTP is saturable and Mg(2+)-dependent. Nucleotides compete with [alpha-32P]GTP binding in the following order: GTP > GDP > Gpp(NH)p > App(NH)p. Dopamine stimulates [alpha-32P]GTP labeling of the 40- and 45-kDa bands. A binding increase of 300-400% is observed at 10 microM dopamine. Isoproterenol (10 microM) stimulates [alpha-32P]GTP binding only to the 45-kDa protein band. The effects of dopamine and isoproterenol are blocked by their respective receptor antagonists, fluphenazine and propranolol. The individual G proteins activated by dopamine are resolved by immunoprecipitation of stimulated [alpha-32P]GTP binding to G alpha s, G alpha i, and G alpha o with specific anti-G alpha antisera. Dopamine stimulates [alpha-32P]GTP binding to G alpha s and G alpha i while the labeling of G alpha o was not significantly changed. Pertussis toxin-mediated ADP ribosylation prevents the activation of G alpha i which is mediated by dopamine receptor stimulation. The methods described are useful in defining the coupling of specific neurotransmitter receptors to specific G proteins in native membranes. These procedures also allow measurements of receptor stimulation of individual G proteins in intact biological membranes.