Molecular Basis for Angiotensin II Receptor Function(s)

描述(由申请人提供)：我们广泛的、长期的目标是了解血管紧张素II受体(Ang II)功能的结构基础。1型(AT1)受体在调节血压、水矿物质平衡、心肌肥大和细胞增殖等方面介导对Ang II的不同细胞内反应。AT1受体是一种新的研究范式，因为它是一种G蛋白偶联受体，除了G蛋白外，它还具有直接激活酪氨酸激酶的独特能力。我们的结构-功能分析已经确定AT1受体与激动剂Ang II和竞争性拮抗剂氯沙坦的关键相互作用。这些研究已经产生了有价值的突变体，具有特定功能的丧失和配体非依赖的激活。用分子模型解释这些突变的结构后果，为AT1受体的激活、脱敏、内化和差异酪氨酸激酶激活的机制提供了重要的见解。一类新的显性-阴性(DN)AT1受体突变体表明，AT1受体的寡聚可能是某些功能所必需的。我们将结合定点突变、定点化学修饰、分子药理学、细胞信号转导和分子模型研究来研究AT1受体结构和功能的一些新方面。 这种竞争性更新的具体目的是：(1)验证基于视紫红质晶体结构的AT1受体同源模型预测的新的结构特征；(2)阐明在治疗上优于竞争性拮抗剂的不可逾越的拮抗剂延长AT1受体抑制的结构基础；以及(3)剖析显性阴性AT1受体突变体的分子基础。具体目标1和2将使用我们以前研究中采用的实验策略。此外，我们将使用定点半胱氨酸修饰研究来获得结构信息。将使用免疫共沉淀和荧光共振能量转移研究来测试突变体的非生产性G蛋白相互作用和寡聚作用。将评估DN突变对Ang-Il特异性细胞功能和蛋白质组反应的影响。该模型的验证对于识别调节AT1受体功能的结构限制至关重要。精细的分子模型将使拮抗剂的未来得到改进。更好地了解不可逾越的拮抗机制将为设计具有更大治疗益处的新型抗高血压、心脏和肾脏保护药物提供洞察力。对反式显性效应的研究将导致寡聚信号转导的新机制。DN突变体可能成为发现与病理生理相关的细胞内信号转导通路的交叉调节的重要工具。

DESCRIPTION (provided by applicant): Our broad, long-term objective is to understand the structural basis of the functions of receptors for angiotensin II (Ang II). The type 1 (AT1) receptor mediates diverse intracellular responses to Ang II in the regulation of blood pressure, hydromineral balance, cardiac hypertrophy and cell proliferation. The AT1 receptor is a novel paradigm for study since it is a G protein-coupled receptor with the unique ability to directly activate, besides G proteins, tyrosine kinases. Our structure-function analysis has identified critical interactions of the AT1 receptor with the agonist Ang II and the competitive antagonist losartan. These studies have yielded valuable mutants with loss of specific function and ligand-independent activation. Interpretation of structural consequences of these mutations by using a molecular model provides important insight into the mechanism of activation, desensitization, internalization and differential tyrosine kinase activation by the AT1 receptor. A novel class of dominant-negative (dn) AT1 receptor mutants have indicated that oligomerization of AT1 receptor may be essential for some functions. A combination of site-directed mutagenesis, site-directed chemical modification, molecular pharmacology, cell signaling and molecular modeling studies will be used to investigate some novel aspects of AT1 receptor structure-function. The specific aims for this competing renewal are: (1) To validate the novel structural features predicted by the homology model of the AT1 receptor based on the crystal structure of rhodopsin; (2) To elucidate the structural basis of prolonged inhibition of the AT1 receptor by insurmountable antagonists which are therapeutically better than competitive antagonists; and (3) To dissect the molecular basis of dominant-negative AT1 receptor mutants. Specific Aims 1 and 2 will use the experimental strategy employed in our previous studies. In addition, we will use site-directed cystein modification studies to obtain structural information. The dnmutants will be tested for non-productive G-protein interaction and oligomerization using co-immunoprecipitation and fluorescence resonance energy transfer studies. The dn-mutant effects on Ang Il-specific cellular function and proteome responses will be evaluated. Validation of the model is critical for identification of structural constraints regulating the functions of the AT1 receptor. A refined molecular model will allow future improvement of antagonists. A better understanding of the mechanism of insurmountable antagonism will provide insight into design of novel anti-hypertensive, cardio- and reno-protective drugs that harbor greater therapeutic benefits. The study of trans-dominant effects will lead to novel mechanisms of signal transduction through oligomerization. The dn-mutants may serve as important tools for discovering cross regulation of intracellular signal-transduction pathways that are relevant to patho-physiology.