CELLULAR SIGNALING OF THE CANNABINOID RECEPTOR

大麻素受体的细胞信号传导

• 批准号: 2646848
• 负责人: BEGONIA Y HO
• 金额: $ 9.66万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-15 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2646848
• 关键词: CHO cells; G protein; adenylate cyclase; antisense nucleic acid; autoradiography; calcium channel; cannabinoid receptor; cannabinoids; complementary DNA; enzyme activity; oligonucleotides; receptor binding; receptor coupling; transfection

DESCRIPTION: (Applicant's Abstract) The cellular signaling mechanisms of the neuronal CB1 and peripheral CB2 cannabinoid receptor will be examined by evaluating the effectors that couple to these two receptors and the G-protein alpha subunits that mediate receptor-effector coupling. Adenylyl cyclase, Ca2+ channels and phospholipase C will be evaluated as the effectors. We will then test the hypothesis that G proteins of the Galpha-i and Galpha-o are mediators for the signalling of the cannabinoid receptor. The hypothesis will be tested by three complementary approaches. 1) The Galpha subunits that are activated upon stimulation of the cannabinoid receptor will be identified by photolabeling with the GTP analog, [alpha-32P]GTP azidoanilide, and subsequent immunoblotting with a panel of anti-Galpha antibodies. 2) The specific Galpha subunits that couple the receptor to the various effectors will be evaluated by using antisense RNA or antisense oligodeoxynucleotides directed against various Galpha subunits. 3) The specific Galpha subunits involved in coupling will then be confirmed with antibodies raised against different Galpha subunits which will interfere with the link in receptor -> G protein coupling. These three approaches will be used in two types of cellular models: NG108, GH4C1 and HL60 cells that express endogenous CB1 and CB2 cannabinoid receptors; and CHO and COS7 cells that express the receptors heterologously. The goal is to test whether there is any specificity in receptor -> G protein -> effector coupling, by comparing the signaling mechanisms of two cannabinoid receptors in different cellular environment.

描述：（申请人摘要） 神经元CB1和外周CB2的细胞信号传导机制 将通过评估效应器来检查大麻素受体 与这两个受体和介导的 G 蛋白 α 亚基偶联 受体-效应器耦合。 腺苷酸环化酶、Ca2+ 通道和 磷脂酶 C 将作为效应子进行评估。 然后我们将测试 假设 Galpha-i 和 Galpha-o 的 G 蛋白是 大麻素受体的信号传导。 假设将被检验 通过三种互补的方法。 1) Galpha 亚基是 刺激大麻素受体后被激活将被识别为 用 GTP 类似物、[α-32P]GTP 叠氮苯胺进行光标记，以及 随后使用一组抗 Galpha 抗体进行免疫印迹。 2) 的 将受体与各种效应器偶联的特定 Galpha 亚基 将使用反义RNA或反义寡脱氧核苷酸进行评估 针对各种 Galpha 亚基。 3) 特定的Galpha亚基 然后用针对该抗体产生的抗体来确认参与偶联 不同的 Galpha 亚基会干扰受体的连接 -> G蛋白偶联。 这三种方法将用于两种类型 细胞模型：表达内源CB1的NG108、GH4C1和HL60细胞 和 CB2 大麻素受体；以及表达 异源受体。 目的是测试是否存在 受体 -> G 蛋白 -> 效应器偶联的特异性，通过比较 不同细胞中两种大麻素受体的信号传导机制 环境。