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.
Friedman
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Department of Psychiatry, Medical College of Pennsylvania, Philadelphia 19129.
Butkerait
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Wang
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D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography / Q000379:methods; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005476:Fluphenazine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000502:physiology*; D011954:Receptors, Dopamine / Q000187:drug effects / Q000502:physiology*
D006150:Guanine Nucleotides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011942:Receptors, Adrenergic, alpha; D011954:Receptors, Dopamine; D000266:Adenylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D011433:Propranolol; D019204:GTP-Binding Proteins; D007545:Isoproterenol; D005476:Fluphenazine; D004298:Dopamine