Crystallization and structure determination of the angiotensin II type 1 receptor

血管紧张素 II 1 型受体的结晶和结构测定

• 批准号: 8166392
• 负责人: Brian K Kobilka
• 金额: $ 28.62万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8166392
• 关键词: Adenosine; Adenylate Cyclase; Adopted; Adrenergic Agents; Affinity; Agonist; Angiotensin II; Angiotensin Receptor; Arrestins; Cardiac; Cardiovascular Diseases; Cardiovascular Physiology; Cell Surface Receptors; Chimeric Proteins; Coupling; Crystallization; Crystallography; Development; Drug Delivery Systems; Drug Design; Exploratory/Developmental Grant; Funding; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Heart failure; Heterotrimeric GTP-Binding Proteins; Human; Hypertension; Hypotension; Imagery; Integral Membrane Protein; Investigation; Ion Channel; Ligand Binding; Ligands; Marketing; Mitogen-Activated Protein Kinases; Molecular; Molecular Conformation; Morbidity - disease rate; Outcome; Performance; Pharmaceutical Preparations; Phospholipase C; Physiological; Physiological Processes; Play; Property; Proteins; Receptor, Angiotensin, Type 1; Regulation; Renin-Angiotensin System; Resolution; Roentgen Rays; Role; Route; Signal Pathway; Signal Transduction; Structure; Therapeutic; United States; Work; adrenergic; arrestin 2; arrestin3; base; high risk; hypertension treatment; hypertensive heart disease; insight; member; mortality; novel; receptor; receptor binding; receptor function; src-Family Kinases; structural biology; therapeutic target

DESCRIPTION (provided by applicant): The goal of this R21 proposal is to obtain a high-resolution crystal structure of the angiotensin II type 1 receptor (AT1R). The AT1R is a member of the Class A G protein coupled receptor (GPCR) superfamily that plays important roles in the regulation of cardiovascular function. Drugs acting on the AT1R are currently used in the treatment of hypertension and heart failure. There are three distinct classes of drugs for the AT1aR: (1) classical antagonists, angiotensin receptor blockers (ARBs) that stabilize the receptor in an inactive conformation, (2) the endogenous agonist angiotensin II that stabilizes an active conformation of the receptor capable of signaling through both the G proteins and the ? -arrestins, and (3) a highly specific ? -arrestin biased agonist that stabilizes a conformation of the receptor that is capable of signaling exclusively through ? -arrestins without any detectable activation of G proteins. Such ??arrestin biased agonists have unique pharmacological and therapeutic properties, distinct from classical agonists or antagonists, e.g. they lower blood pressure (like antagonists) but increase cardiac performance (like agonists). Little is known about the structural basis by which these different types of ligands regulate receptor function. We propose to begin a detailed investigation of the structural biology of the AT1aR through an R21 mechanism. Our goal for this proposal is to demonstrate that we can obtain diffraction quality crystals and a high-resolution structure of the AT1R bound to a high-affinity antagonist. The proposed work is high-risk and high-impact. If successful, the outcome of this R21 proposal will enable us to obtain funding for a more thorough structural characterization of the AT1aR in different conformational states. The long-term objective of this proposal is to determine the high-resolution crystal structures of the AT1aR in three different conformations; the inactive conformation stabilized by an ARB; the classical active conformation stabilized by angiotensin II; and an active conformation capable of signaling through only 2- arrestins stabilized by a ? -arrestin biased agonist. These structures will facilitate the development of safer and more effective therapeutics for heart failure and hypertension. Specific Aims include: 1) Generate an AT1aR-T4lysozyme fusion protein and adjust the linkers between these two proteins to optimize AT1aR functional expression and stability. 2) Establish conditions for expression and purification of AT1aR for crystallography trials. 3) Crystallize and determine the X-ray crystal structure of the AT1aR. PUBLIC HEALTH RELEVANCE: Hypertension and heart failure represent major causes of morbidity and mortality in the United States. The renin-angiotensin system plays a central role in regulating cardiovascular function, and the angiotensin receptor is an important therapeutic target for cardiovascular disease. Successful completion of the aims of this proposal will facilitate the development of more selective and effective drugs for heart failure and hypertension.

描述（由申请人提供）：本R21提案的目标是获得血管紧张素II 1型受体（AT 1 R）的高分辨率晶体结构。AT 1 R是A类G蛋白偶联受体（GPCR）超家族的成员，在心血管功能的调节中发挥重要作用。作用于AT 1 R的药物目前用于治疗高血压和心力衰竭。有三种不同类型的药物AT 1aR：（1）经典的拮抗剂，血管紧张素受体阻滞剂（ARBs），稳定受体在一个非活性构象，（2）内源性激动剂血管紧张素II，稳定的活性构象的受体能够通过两个G蛋白和？- 抑制素，以及（3）高度特异性？-arrestin偏向激动剂，稳定的构象的受体，能够通过信号只？- 没有任何可检测的G蛋白活化的抑制蛋白。什么？？抑制蛋白偏向性激动剂具有独特的药理学和治疗性质，不同于经典的激动剂或拮抗剂，例如它们降低血压（类似于拮抗剂）但增加心脏性能（类似于激动剂）。这些不同类型的配体调节受体功能的结构基础知之甚少。我们建议通过R21机制开始对AT 1aR的结构生物学进行详细研究。我们的目标是证明我们可以获得衍射质量的晶体和高分辨率的结构的AT 1 R结合到一个高亲和力的拮抗剂。拟议的工作是高风险和高影响的。如果成功的话，R21提案的结果将使我们能够获得资金，对不同构象状态下的AT 1aR进行更彻底的结构表征。这项建议的长期目标是确定高分辨率的晶体结构的AT 1aR在三种不同的构象;非活性构象稳定的ARB;经典的活性构象稳定的血管紧张素II;和一个积极的构象能够通过信号只有2- arrestins稳定的？- 抑制蛋白偏向激动剂。这些结构将有助于开发更安全和更有效的心力衰竭和高血压治疗方法。具体目标包括：1）制备AT 1aR-T4溶菌酶融合蛋白，并调整两蛋白之间的接头以优化AT 1aR的功能表达和稳定性。2)为晶体学试验建立AT 1aR的表达和纯化条件。3)结晶并确定AT 1aR的X射线晶体结构。 公共卫生相关性：高血压和心力衰竭是美国发病率和死亡率的主要原因。肾素-血管紧张素系统在调节心血管功能中起重要作用，血管紧张素受体是心血管疾病的重要治疗靶点。成功完成本提案的目标将有助于开发更具选择性和更有效的心力衰竭和高血压药物。