Cannabinoid receptor stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding in rat brain membranes

Life Sci. 1996;59(8):659-68. doi: 10.1016/0024-3205(96)00347-5.


Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [35S]GTP gamma S binding in rat brain membranes. Assays were conducted with 0.05 nM [35S]GTP gamma S, incubated with rat cerebellar membranes, 1-30 microM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [35S]GTP gamma S binding increased with increasing concentrations of GDP, with 10-30 microM GDP providing approximately 150-200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [35S]GTP gamma S binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [35S]GTP gamma S binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [35S]GTP gamma S binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [35S]GTP gamma S autoradiography. These results demonstrate that [35S]GTP gamma S binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.

Publication types

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

MeSH terms

  • Animals
  • Autoradiography
  • Benzoxazines
  • Binding Sites
  • Cannabinoids / agonists
  • Cannabinoids / metabolism
  • Cannabinoids / pharmacology
  • Cell Membrane / metabolism
  • Cerebellum / metabolism*
  • GTP-Binding Proteins / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism*
  • Guanosine Diphosphate / pharmacology
  • Kinetics
  • Male
  • Morpholines / pharmacology
  • Naphthalenes / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, Cannabinoid
  • Receptors, Drug / drug effects
  • Receptors, Drug / physiology*
  • Sulfur Radioisotopes


  • Benzoxazines
  • Cannabinoids
  • Morpholines
  • Naphthalenes
  • Receptors, Cannabinoid
  • Receptors, Drug
  • Sulfur Radioisotopes
  • Guanosine Diphosphate
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • (3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone
  • GTP-Binding Proteins