RECEPTOR-MEDIATED INHIBITION OF ADENYLATE CYCLASE

受体介导的腺苷酸环化酶抑制

• 批准号: 3287530
• 负责人: THOMAS E COTE
• 金额: $ 5.5万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287530
• 关键词: ADP ribosylation; G protein; adenosine diphosphate; adenylate cyclase; alpha adrenergic receptor; binding proteins; chemical structure function; dopamine receptor; drug metabolism; enzyme inhibitors; enzyme mechanism; enzyme structure; guanosine triphosphate; guanosinetriphosphatases; laboratory rat; muscarinic receptor; neuropeptide receptor; neuropharmacology; neurotransmitters; nickel; opioid receptor; pertussis toxin

The long term objective of this research plan is to gain insight into the mechanism of action of receptors inhibiting adenylate cyclase activity. Numerous neuronal receptors including opiate, D-2 dopaminergic, alpha2-adrenergic, purinergic, and cholinergic are believed to modulate neuronal activity by inhibiting adenylate cyclase. Since these receptors play a critical role in modulating the functioning of the brain, insight into their mechanism of action is fundamental to the understanding and treatment of neurological disorders involving these receptors. Recently, a Mu-opiate receptor with pharmacological properties similar to brain Mu-opiate receptors was discovered on the cells of a prolactin secreting tumor termed 7315c. Preliminary results suggest that this receptor is associated with the inhibitory GTP binding protein, termed Ni, which acts as a transducer between the inhibitory receptor and adenylate cyclase. The specific aim of this research proposal is to determine how the Mu-opiate receptor enhances the interaction of guanyl nucleotides with Ni. It is hypothesized that activation of the Mu-opiate receptor enhances the exchange of GDP for GTP at Ni. Initially, opiates will be tested for their ability to stimulate GTPase activity in 7315c membranes. Next, GDP will be tested for its ability to block both the Gpp(NH)p- and the GTP-induced activation of Ni. Opiate agonists will also be tested for their ability to enhance the removal of Gpp(NH)p from Ni and cause predictable changes in adenylate cyclase activity. Pertussis toxin has recently been proposed to induce an ADP ribosylation of Ni resulting in the uncoupling of Ni and inhibitory receptors. Pertussis toxin will be tested for its ability to block Mu-opiate receptor-mediated exchange of guanyl nucleotides at Ni. It will also be determined if pertussis toxin has a direct effect on inhibitory receptors. Intermediate lobe membranes containing an inhibitory D-2 dopamine receptor will be treated with pertussis toxin and then fused with 7315c membranes (which contain no D-2 receptor). The D-2 dopamine receptor will then be tested for its ability to recouple with Ni and inhibit adenylate cyclase. Finally, an opiate affinity column will be used in an attempt to isolate the Mu-opiate receptor in close association with Ni.

该研究计划的长期目标是深入了解 受体抑制腺苷酸环化酶活性的作用机制。 许多神经元受体，包括阿片类、D-2 多巴胺能受体、 α2-肾上腺素能、嘌呤能和胆碱能被认为可以调节 通过抑制腺苷酸环化酶来抑制神经元活性。 由于这些受体 在调节大脑功能、洞察力方面发挥关键作用 深入了解其作用机制是理解和 治疗涉及这些受体的神经系统疾病。 最近，一个 Mu-鸦片受体具有与脑相似的药理特性 在分泌催乳素的细胞上发现了 Mu-阿片受体 肿瘤称为7315c。 初步结果表明该受体是 与称为 Ni 的抑制性 GTP 结合蛋白相关，其作用 作为抑制性受体和腺苷酸环化酶之间的转导器。 这 本研究计划的具体目的是确定 Mu-阿片类药物如何 受体增强鸟苷酸与Ni的相互作用。 这是 假设 Mu-阿片受体的激活增强了 GDP 与 GTP 的兑换为 Ni。 最初，阿片类药物将接受测试 刺激 7315c 膜中 GTP 酶活性的能力。 接下来，GDP将是 测试了其阻断 Gpp(NH)p 和 GTP 诱导的能力 Ni的活化。 阿片激动剂的能力也将受到测试 增强从 Ni 中去除 Gpp(NH)p 并引起可预测的变化 腺苷酸环化酶活性。 最近有人提出百日咳毒素 诱导 Ni 的 ADP 核糖基化，导致 Ni 和 抑制性受体。 将测试百日咳毒素的能力 阻断 Mu-阿片受体介导的 Ni 处鸟苷酸交换。 它 还将确定百日咳毒素是否对 抑制性受体。 中叶膜含有抑制物 D-2多巴胺受体会用百日咳毒素处理然后融合 带有 7315c 膜（不含 D-2 受体）。 D-2 多巴胺 然后将测试受体与 Ni 重新偶联的能力 抑制腺苷酸环化酶。 最后，将使用阿片亲和柱 试图分离与密切相关的 Mu-阿片受体 尼。