CANNABINOID AGONIST REGULATION OF SIGNAL TRANSDUCTION

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DESCRIPTION(Adapted from applicant's abstract): This is a new application from an investigator who currently holds an R29 Award, but has never had an RO1 award. He proposes three specific aims to establish the multiplicity of coupling of cannabinoid (CB) receptors with G proteins and to determine which G proteins mediate downstream signaling in brain. In specific aim #1, he will investigate the interaction of CB1 receptors in rat brain with specific G-alpha subunits in response to full, partial and inverse cannabinoid agonists derived from four structural classes. He will use a newly developed elegant technique in which receptor-activated G protein autoradiography is performed using brain sections with a GTP photoaffinity ligand. The studies in the brain slices will be augmented by isolating key areas from each slice, solubilizing the sections and determining which G proteins are specifically activated by high resolution gel techniques. In some cases, the gel techniques will be augmented by immunoprecipitations with specific G protein antibodies. Full concentration-effect curves will be employed using membranes prepared from those specific brain regions. The second specific aim is to correlate CB ligand-selective G protein regulation with specific effects or coupling in cerebellar granular cells. He will use cerebellar granular cells as primary neuronal cultures to compare the potency and efficacy of selected full, partial and inverse cannabinoid agonists to activate specific G proteins in the membrane preparations. He will next determine which pertussis-toxin sensitive intracellular effectors are regulated by CB1 receptors in those cells. The effectors to be evaluated will include the inhibition of adenylyl cyclase activity, inhibition of voltage-dependent calcium currents, activation of MAPK activity and finally, activation of inwardly rectifying potassium channels. The third portion of this aim involves identifying the specific G protein alpha subunits responsible for the coupling to those effectors by employing antisense oligonucleotides directed against specific G protein alpha subunits. He will also determine the correlation between the potency and efficacy of different CB ligands to coupled individual G proteins and regulates the distinct intracellular effectors. The third specific aim is to investigate potential mechanisms underlying CB agonist specific trafficking of responses in CB1 receptor transfected C6 glioma cells. The hypothesis to be tested here is that the ratio of CB1 receptors to G proteins within cells will contribute to the ability of different agonists to selectively traffic responses. He will transfect C6 glioma cells with the cDNA's encoding CB1 receptors to generate clones expressing a wide range of receptor densities. He will then compare the potency and efficacy of selected full, partial and inverse CB agonist to activate specific G proteins and effectors as a function of receptor density.