CANNABINOID AGONIST REGULATION OF SIGNAL TRANSDUCTION

描述（改编自申请人摘要）： 这是一个新的应用程序从一个研究员谁目前持有R29 但从未获得过RO 1奖。他提出了三个具体目标， 建立大麻素（CB）受体与G 蛋白质，并确定哪些G蛋白介导下游信号传导， 个脑袋在具体目标#1中，他将研究CB 1受体的相互作用， 在大鼠脑中，特异性G-α亚单位对完全、部分和 衍生自四种结构类别的反向大麻素激动剂。他将利用 一种新开发的优雅技术，其中受体活化G蛋白 使用具有GTP光亲和性的脑切片进行放射自显影 配体。大脑切片的研究将通过分离密钥来增强 从每个切片的面积，溶解切片，并确定哪些G 蛋白质通过高分辨率凝胶技术被特异性激活。在一些 在某些情况下，凝胶技术将通过免疫沉淀增强， 特异性G蛋白抗体完整的浓度-效应曲线如下： 使用从这些特定的大脑区域制备的膜。 第二个具体目标是将CB配体选择性G蛋白 在小脑颗粒细胞中具有特定效应或耦合的调节。他 将使用小脑颗粒细胞作为原代神经元培养物， 选择的完全、部分和反向大麻素激动剂的效力和功效 来激活膜制备物中的特定G蛋白。接下来他将 确定哪些百日咳毒素敏感的细胞内效应物受到调节 通过这些细胞中的CB 1受体。要评估的效应器将包括 腺苷酸环化酶活性抑制，电压依赖性 钙电流，激活MAPK活性，最后，激活 内向整流钾通道这一目标的第三部分涉及 鉴定负责偶联的特定G蛋白α亚基 这些效应物通过使用针对 特异性G蛋白α亚单位。他还将确定 不同CB配体对偶联个体的效力和功效之间的差异 G蛋白和调节不同的细胞内效应。 第三个具体的目标是调查潜在的机制， CB 1受体转染的C6胶质瘤中激动剂特异性运输反应 细胞这里要检验的假设是，CB 1受体与G 细胞内的蛋白质将有助于不同激动剂的能力， 选择性地进行交通响应。他将用免疫荧光法检测C6神经胶质瘤细胞。 编码CB 1受体的cDNA，以产生表达广泛的 受体密度然后，他将比较所选药物的效力和功效， 完全、部分和反向CB激动剂，以激活特异性G蛋白， 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.