B-ADRENERGIC RECEPTOR STUCTURE & DESENSITIZATION

B-肾上腺素能受体结构

• 批准号: 3279136
• 负责人: RICHARD B CLARK
• 金额: $ 13.32万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279136
• 关键词: adenylate cyclase; beta adrenergic receptor; cell free system; cyclic AMP; diterpenes; enzyme induction /repression; epinephrine; hormone receptor; hormone regulation /control mechanism; molecular cloning; neoplastic cell culture for noncancer research; phosphorylation; prostaglandin E; protein kinase; tissue /cell culture

The intent of this proposal is to test our hypothesis that cAmp- dependent protein kinase (PKA) mediates the rapid physiological heterologous desensitization of hormonal stimulation of adenylate cyclase in the S49 wild type (WT) lymphoma cell line. Previous work had demonstrated only a homologous desensitization in response to relatively high hormone concentrations, which was independent of Gs' cAMP, and PKA. We have recently found that cAMP activation of PKA plays a crucial role in the heterologous desensitization of the beta-adrenergic receptor (beta-AR) and the prostaglandin E1 (PGE1) receptor in WT cells in response to physiological concentr- ations of epinephrine or PGE1 (0-20 nM). Use of Mg2+ levels in our adenylate cyclase assays which mimic intracellular concentrations (0.1-0.5 mM) revealed: 1) heterologous desensitization of the WT adenylate cyclase, but not that of cyc or kin cells, and 2) that addition of dibutyryl cAMP along with epinephrine to the cyc cells caused heterologous desensitization. We will pursue a genetic-biochemical approach using the S49 WT and mutant cells to carry out the following specific aims: 1) characterization of the heterologous desensitization of the WT cells and derived clones deficient in either adenylate cyclase or PKA in response to the treatment of these cells with 0-20 nM PGE1 or epinephrine, with cAMP analogues and forskolin, and with the hormones in combination with these agents; 2) elucidation of conditions for cell-free heterologous desensitization of adenylate cyclase, and use of this system to examine the effects of purified PKA and phosphatases on the phosphorylation-dephosphorylation of the beta-AR and the correlation with heterologous desensitization- resensitization; 3) characterization of the phosphorylation of the beta-AR in response to treatment of intact cells with low concentrations of hormones and forskolin; and 4) determination of the generality of the role of PKA in the heterologous desensitization of receptors which stimulate adenylate cyclase in mammalian cell lines.

这项提议的目的是测试我们的假设，即营地- 依赖蛋白激酶(PKA)介导的快速生理性 腺苷类激素刺激的异种脱敏作用 S49野生型(WT)淋巴瘤细胞系中的环化酶。上一首 研究表明，只有一种相应的脱敏反应 到相对较高的激素浓度，这是独立的 GS的营地和PKA。我们最近发现，cAMP激活 在异源脱敏中起关键作用 β-肾上腺素能受体与前列腺素E_1 WT细胞(PGE1)受体对生理浓度的反应 肾上腺素或前列腺素E_1(0-20 NM)。镁离子水平在中国的应用 我们的腺苷酸环化酶检测模拟细胞内 浓度(0.1-0.5 mM)显示：1)异源 WT腺苷环化酶的脱敏作用，但不是Cyc的脱敏作用 或亲缘细胞，以及2)添加二丁酰cAMP以及 肾上腺素对Cyc细胞有异源脱敏作用。 我们将使用S49 WT进行遗传生化方法和 突变细胞实现以下具体目标：1) WT异源脱敏作用的表征 腺苷环化酶缺陷的细胞和衍生克隆 PKA对0-20 nM PGE1处理这些细胞的反应 或肾上腺素，与cAMP类似物和Forsklin，以及与 激素与这些药物的结合；2)阐明 腺苷脱敏的无细胞异源脱敏条件 并用此系统检测纯化后的效果 蛋白酪氨酸酶和磷酸酶在细胞的磷酸化-去磷酸化过程中的作用 β-AR及其与异源脱敏的相关性 再敏化；3)磷酸化的表征 β-AR对低密度脂蛋白处理完整细胞的反应 激素和福斯可林的浓度；以及4)测定 PKA在异源生物中作用的概括性 激活腺苷环化酶的受体的脱敏作用 哺乳动物细胞系。