ANGIOTENSIN II RECEPTORS AND SIGNALING MECHANISMS

血管紧张素 II 受体和信号传导机制

• 批准号: 6107983
• 负责人: Kevin J Catt
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6107983
• 关键词: adrenal glands; adrenal medulla; angiotensin II; biological signal transduction; calcineurin; calcium flux; enzyme activity; gene expression; hormone receptor; hormone regulation /control mechanism; isozymes; neurohormones; phosphatidylinositols; phosphotransferases; protein structure function; receptor binding; receptor coupling; receptor expression; tissue /cell culture

This research project addresses the structure-function properties and signaling pathways of the AT1 receptor, which mediates the physiological actions of angiotensin II (Ang II) in the cardiovascular system, adrenal, kidney, brain, liver, and other target tissues. In addition to its specific roles in Ang II target cells, the AT1 receptor serves as a model for the function of other G protein coupled receptors (GPCRs). Our analysis of the roles of specific amino acids in AT1 receptor function has identified residues involved in ligand binding, receptor activation, G protein coupling, and receptor internalization. Recent examples include the importance of two seventh transmembrane domain asparagine, Asn294 and Asn295, in receptor activation and ligand binding, respectively. Also, membrane-proximal amino acids in the carboxyterminal cytoplasmic domain, in particular Phe301, were found to be significant determinants of receptor expression at the cell surface. Some of the functions of GPCRs known to be regulated by agonist-induced phosphorylation of serine/threonine residues in the cytoplasmic tail and/or intracellular loops of the receptor. A highly specific antibody to the AT1 receptor was raised against a fusion protein containing the 92-amino acid C-terminal fragment of the receptor. This antibody permitted the analysis of agonist-induced phosphorylation in the native AT1 receptor immunoprecipitated from Ang II-treated bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, and was correlated with the degree of ligand occupancy of the receptor. The AT1 receptor was also phosphorylated by activation of protein kinases A and C, but to a lesser extent than that induced by Ang II. Inhibition of PKC activity by stauresporine abolished TPA-induced receptor phosphorylation but enhanced Ang II-induced phosphorylation. This suggests that PKC may exert an inhibitory action on the G protein receptor kinases that are regarded as the major mediators of agonist-induced receptor phosphorylation. An investigation of the sites at which the AT1 receptor is phosphorylated was performed on mutant receptors bearing deletions or mutations in the cytoplasmic tail. This study utilized epitope-tagged AT1 receptors that were expressed in COS cells and immunoprecipitated with an anti-HA antibody after agonist stimulation. The Ang II-induced phosphorylation of the AT1 receptor was found to be confined to an 11-amino acid serine/threonine-rich region in the C-terminal cytoplasmic tail, indicating the importance of this region in receptor internalization and

该研究项目解决了 AT1的结构功能和信号通路 受体，介导血管紧张素II的生理作用 （ANG II）在心血管系统中，肾上腺，肾脏，大脑，肝脏， 和其他靶组织。除了其在Ang II中的特定作用外 靶细胞，AT1受体作为功能的模型 其他G蛋白偶联受体（GPCR）。我们对 特异性氨基酸在AT1受体功能中的作用已确定 参与配体结合，受体激活，G蛋白的残基 耦合和受体内在化。最近的例子包括 两个第七跨膜域天冬酰胺的重要性， ASN294和ASN295，在受体激活和配体结合中， 分别。此外，膜中的膜氨基酸 羧基细胞质结构域，特别是PHE301 发现是受体表达的重要决定因素 细胞表面。 GPCR的某些功能已知 由激动剂诱导的丝氨酸/苏氨酸的磷酸化调节 细胞质尾巴中的残留物和/或细胞内环的残留物 受体。提高了对AT1受体的高度特异性抗体 针对含有92个氨基酸C末端的融合蛋白 受体的碎片。该抗体允许分析 激动剂诱导的天然AT1受体的磷酸化 从ANG II处理的牛肾上腺免疫沉淀 肾小球细胞。受体磷酸化很快，是 与受体的配体占用程度相关。这 AT1受体还通过蛋白的激活而被磷酸化 激酶A和C，但比Ang II引起的较小程度较小。 通过静态孢氨酸酯抑制PKC活性消除了TPA诱导的 受体磷酸化，但增强了ANG II诱导的 磷酸化。这表明PKC可能会施加抑制作用 对G蛋白受体激酶的作用，被视为 激动剂诱导的受体磷酸化的主要介体。一个 研究AT1受体的位点 在轴承的突变受体上进行磷酸化 细胞质尾部的缺失或突变。这项研究使用了 在COS细胞中表达的表位标记的AT1受体和 用激动剂后用抗HA抗体免疫沉淀 刺激。 ANG II诱导的AT1磷酸化 发现受体局限于11-氨基酸 C末端细胞质尾巴中的丝氨酸/苏氨酸富含区域， 表明该区域在受体内在化中的重要性 和