CANNABINOID AGONIST REGULATION OF SIGNAL TRANSDUCTION

大麻素激动剂对信号转导的调节

基本信息

批准号：
6628361
负责人：
Paul L Prather
金额：
$ 18.09万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-01 至 2005-01-31
项目状态：
已结题

项目摘要

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.

描述（改编自申请人的摘要）：这是目前持有 R29 的调查员的新申请奖，但从未获得过RO1奖。他提出了三个具体目标建立大麻素 (CB) 受体与 G 的偶联多重性蛋白并确定哪些 G 蛋白介导下游信号传导脑。在具体目标#1中，他将研究 CB1 受体的相互作用在具有特定 G-α 亚基的大鼠大脑中，对完全、部分和源自四个结构类别的反向大麻素激动剂。他会用一种新开发的优雅技术，其中受体激活的 G 蛋白使用具有 GTP 光亲和力的脑切片进行放射自显影配体。脑切片研究将通过隔离关键信息得到加强每个切片的区域，溶解切片并确定哪个 G 蛋白质通过高分辨率凝胶技术被特异性激活。在一些在这种情况下，凝胶技术将通过免疫沉淀得到增强特异性G蛋白抗体。完整的浓度效应曲线将是使用由这些特定大脑区域制备的膜。第二个具体目标是关联 CB 配体选择性 G 蛋白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来比较选定的完全、部分和反向大麻素激动剂的效力和功效激活膜制剂中的特定 G 蛋白。他接下来将确定哪些百日咳毒素敏感的细胞内效应器受到调节通过这些细胞中的 CB1 受体。要评估的效应器将包括抑制腺苷酸环化酶活性，抑制电压依赖性钙电流，MAPK 活性的激活，最后，内向整流钾通道。该目标的第三部分涉及识别负责偶联的特定 G 蛋白 α 亚基通过使用针对这些效应子的反义寡核苷酸特定的G蛋白α亚基。他还将确定相关性不同 CB 配体对偶联个体的效力和功效之间的关系 G 蛋白并调节不同的细胞内效应器。第三个具体目标是研究 CB 的潜在机制 CB1受体转染的C6神经胶质瘤中激动剂特异性反应运输细胞。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有选择地进行流量响应。他将用编码 CB1 受体的 cDNA 可生成表达多种受体的克隆受体密度。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，可激活特定的 G 蛋白和效应器作为受体密度的函数。