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：（申请人的摘要） 神经元CB1和周围CB2的细胞信号传导机制 大麻素受体将通过评估效应子来检查 将这两个受体和介导的G蛋白α亚基求助 受体效应耦合。 腺苷酸环化酶，Ca2+通道和 磷脂酶C将被评估为效应子。 然后我们将测试 假设Galpha-I和Galpha-O的G蛋白是介体 大麻素受体的信号传导。 该假设将进行检验 通过三种补充方法。 1）Galpha亚基是 刺激后大麻素受体被激活将被鉴定 使用GTP类似物[alpha-32p] GTP氮杂苯胺和 随后使用一系列抗Galpha抗体进行免疫印迹。 2） 将受体与各种效应子搭配的特定galpha亚基 将通过使用反义RNA或反义寡脱氧核苷酸来评估 针对各种Galpha亚基。 3）特定的galpha亚基 然后，辅助涉及的抗体将确认 不同的galpha亚基将干扰受体中的链接 - > G蛋白偶联。 这三种方法将用于两种类型的 细胞模型：表达内源性CB1的NG108，GH4C1和HL60细胞 和CB2大麻素受体；以及表达的CHO和COS7单元 受体异源。 目标是测试是否有 通过比较受体 - > g蛋白 - >效应子偶联的特异性 不同细胞中两个大麻素受体的信号传导机制 环境。