B-ADRENERGIC RECEPTOR STUCTURE & DESENSITIZATION

B-肾上腺素能受体结构

• 批准号: 3279141
• 负责人: RICHARD B CLARK
• 金额: $ 17.83万
• 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/3279141
• 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.

这项提议的目的是检验我们的假设，即casino- 依赖性蛋白激酶（PKA）介导快速的生理 腺苷酸激素刺激的异源脱敏 在S49野生型（WT）淋巴瘤细胞系中的环化酶。 先前 工作证明，只有一个同源的脱敏反应， 相对较高的激素浓度，这是独立的 Gs的cAMP和PKA。 我们最近发现cAMP激活 PKA在异源脱敏中起着至关重要的作用， β-肾上腺素能受体（β-AR）和前列腺素E1 （PGE 1）受体在野生型细胞中响应生理浓度- 肾上腺素或PGE 1（0-20 nM）。 使用Mg 2+水平 我们的腺苷酸环化酶试验模拟细胞内 浓度（0.1-0.5 mM）显示：1）异源 WT腺苷酸环化酶的脱敏，而不是cyc 或皮肤细胞，和2）添加二丁酰cAMP沿着 肾上腺素对cyc细胞引起异源性脱敏。 我们将使用S49 WT进行遗传生化方法， 突变细胞进行以下具体目标：1） WT的异源脱敏的表征 细胞和衍生的克隆缺乏腺苷酸环化酶或 PKA对用0-20 nM PGE 1处理这些细胞的响应 或肾上腺素，与cAMP类似物和毛喉素，并与 激素与这些药物的组合; 2）阐明 腺苷酸的无细胞异源脱敏的条件 环化酶的作用，并使用该系统来检查纯化的环化酶的作用。 蛋白激酶A和磷酸酶对蛋白磷酸化-去磷酸化的影响 β-AR及其与异源脱敏的相关性 再敏化; 3）表征的磷酸化的 β-AR对用低浓度 激素和毛喉素的浓度;和4）测定 PKA在异源细胞中作用的一般性， 刺激腺苷酸环化酶的受体的脱敏 哺乳动物细胞系。