CANNABINOID AGONIST REGULATION OF SIGNAL TRANSDUCTION

描述(改编自申请人的摘要)： 这是一名调查员的新申请，他目前持有R29 获奖，但从未获得过RO1奖。他提出了三个具体目标，以 建立大麻素(CB)受体与G偶联的多样性 并确定哪些G蛋白介导了下游信号转导 大脑。在特定的目标1中，他将研究CB1受体的相互作用 大鼠脑内特定G-α亚基对完全、部分和 来自四个结构类别的反向大麻素激动剂。他将使用 一种新开发的优雅技术，其中受体激活的G蛋白 放射自显影是使用具有GTP光亲和力的大脑切片进行的 莱兰德。大脑切片的研究将通过分离关键字来加强 每个切片的面积，溶解各部分并确定哪个G 蛋白质通过高分辨率凝胶技术被特异性地激活。在一些 在这种情况下，凝胶技术将通过免疫沉淀得到加强 特异性G蛋白抗体。完整的浓度-效应曲线将 使用从这些特定大脑区域制备的膜。 第二个特定目的是关联CB配体选择G蛋白 在小脑颗粒细胞中具有特定作用或偶联的调节。他 将使用小脑颗粒细胞作为原代神经元培养细胞来比较 选定的完全、部分和反向大麻素激动剂的效力和疗效 激活膜制剂中的特定G蛋白。他将是下一个 确定哪些对百日咳毒素敏感的细胞内效应物受到调控 通过这些细胞中的CB1受体。要评估的效应器将包括 抑制腺酰环化酶活性，抑制电压依赖性 钙电流，激活MAPK活性，最终激活MAPK 向内整流钾通道。这一目标的第三部分涉及 确定负责偶联的特定G蛋白α亚基 通过使用针对这些效应器的反义寡核苷酸 特定的G蛋白α亚基。他还将确定两者之间的关联 不同CB配体对偶联个体效价的比较 G蛋白，并调节不同的细胞内效应器。 第三个具体目标是调查CB潜在的机制 激动剂特异性转运CB1受体在C6神经胶质瘤中的作用 细胞。这里要检验的假设是CB1受体与G的比率 细胞内的蛋白质将有助于不同激动剂的能力 选择性的交通响应。他将用该基因转染人C6胶质瘤细胞 编码CB1受体的cDNAs克隆，表达广泛的 受体密度。然后，他将比较选定的药物的效力和疗效 完全、部分和反向CB激动剂激活特定的G蛋白和 效应器作为受体密度的函数。

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.