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: 8302319
• 负责人: Brian K Kobilka
• 金额: $ 15.75万
• 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/8302319
• 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.

描述（由申请人提供）：该 R21 提案的目标是获得血管紧张素 II 1 型受体 (AT1R) 的高分辨率晶体结构。 AT1R 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AT1R的药物目前用于治疗高血压和心力衰竭。 AT1aR 药物分为三类：(1) 经典拮抗剂、血管紧张素受体阻滞剂 (ARB)，可将受体稳定在非活性构象；(2) 内源性激动剂血管紧张素 II，可稳定受体的活性构象，能够通过 G 蛋白和 ? -arrestins，以及（3）高度特异性的？ -arrestin 偏向激动剂，稳定能够仅通过 ? 发出信号的受体构象。 -arrestins，没有任何可检测到的 G 蛋白激活。这种抑制蛋白偏向激动剂具有独特的药理学和治疗特性，不同于经典的激动剂或拮抗剂，例如它们可以降低血压（如拮抗剂），但可以提高心脏功能（如激动剂）。人们对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 AT1aR 的结构生物学进行详细研究。我们此提案的目标是证明我们可以获得衍射质量的晶体和与高亲和力拮抗剂结合的 AT1R 的高分辨率结构。拟议的工作具有高风险和高影响力。如果成功，该 R21 提案的结果将使我们能够获得资金，以对不同构象状态下的 AT1aR 进行更彻底的结构表征。该提案的长期目标是确定 AT1aR 三种不同构象的高分辨率晶体结构；由 ARB 稳定的非活性构象；血管紧张素 II 稳定的经典活性构象；以及能够仅通过由 ? 稳定的 2-抑制蛋白发出信号的活性构象。 -arrestin偏向激动剂。这些结构将有助于开发更安全、更有效的心力衰竭和高血压疗法。具体目标包括： 1) 生成 AT1aR-T4 溶菌酶融合蛋白并调整这两种蛋白之间的接头以优化 AT1aR 功能表达和稳定性。 2) 建立用于晶体学试验的 AT1aR 表达和纯化条件。 3) 结晶并测定AT1aR的X射线晶体结构。