A method to study [α-32P]GTP binding to the α subunit of GTP-binding proteins in rat brain membranes is described. This method measures receptor-stimulated GTP binding to individual α 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 α subunits of Gi/Go and Gs, respectively. Binding of [α-32P]GTP is saturable and Mg2+-dependent. Nucleotides compete with [α-32P]GTP binding in the following order: GTP > GDP > Gpp(NH)p > App(NH)p. Dopamine stimulates [α-32P]GTP labeling of the 40- and 45-kDa bands. A binding increase of 300-400% is observed at 10 μM dopamine. Isoproterenol (10 μM) stimulates [α-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 [α-32P]GTP binding to Gαs, Gαi, and Gαo with specific anti-Gα antisera. Dopamine stimulates [α-32P]GTP binding to Gαs and Gαi while the labeling of Gαo was not significantly changed. Pertussis toxin-mediated ADP ribosylation prevents the activation of Gαi 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. © 1993 Academic Press, Inc.
