Analysis of Receptor-Stimulated and Basal Guanine Nucleotide Binding to Membrane G Proteins by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Anal Biochem. 1993 Oct;214(1):171-8. doi: 10.1006/abio.1993.1473.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenylyl Imidodiphosphate / pharmacology
  • Animals
  • Autoradiography / methods
  • Binding, Competitive
  • Brain / metabolism*
  • Cell Membrane / metabolism
  • Dopamine / pharmacology
  • Electrophoresis, Polyacrylamide Gel / methods
  • Fluphenazine / pharmacology
  • GTP-Binding Proteins / isolation & purification
  • GTP-Binding Proteins / metabolism*
  • Guanine Nucleotides / pharmacology
  • Guanosine Triphosphate / metabolism*
  • Isoproterenol / pharmacology
  • Kinetics
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism*
  • Phosphorus Radioisotopes
  • Propranolol / pharmacology
  • Rats
  • Receptors, Adrenergic, alpha / drug effects
  • Receptors, Adrenergic, alpha / physiology*
  • Receptors, Dopamine / drug effects
  • Receptors, Dopamine / physiology*

Substances

  • Guanine Nucleotides
  • Phosphoproteins
  • Phosphorus Radioisotopes
  • Receptors, Adrenergic, alpha
  • Receptors, Dopamine
  • Adenylyl Imidodiphosphate
  • Guanosine Triphosphate
  • Propranolol
  • GTP-Binding Proteins
  • Isoproterenol
  • Fluphenazine
  • Dopamine