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; 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）：该R21提案的目标是获得血管紧张素II型1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起着重要作用。目前，作用于AT1R的药物用于治疗高血压和心力衰竭。 AT1AR有三种不同类别的药物：（1）经典拮抗剂，血管紧张素受体阻滞剂（ARB）稳定受体在非活性构象中的稳定，（2）内源性激动剂血管紧张素II，稳定了通过G蛋白和G蛋白和G蛋白和G蛋白和G蛋白和G蛋白的活性构象稳定的？ -arrestins，（3）高度具体？ - 雌激素的偏向激动剂稳定能够仅通过信号传导的受体的构象？ -RARRESTIN，没有G蛋白的任何可检测到的激活。这样的阻碍素有偏的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，例如它们降低了血压（如拮抗剂），但会增加心脏表现（例如激动剂）。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AT1AR的结构生物学进行详细研究。该提案的目标是证明我们可以获得与高亲和力拮抗剂结合的AT1R的衍射质量晶体和高分辨率结构。拟议的工作是高风险和高影响力。如果成功，则该R21提案的结果将使我们能够获得资金，以在不同构象状态下对AT1AR进行更彻底的结构表征。该提议的长期目标是确定三种不同构象中AT1AR的高分辨率晶体结构。 ARB稳定的无活性构象；血管紧张素II稳定的经典活动构象；和一种活跃的构象，能够通过A稳定的2个逮捕蛋白发出信号？ -arrestin偏见的激动剂。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的治疗疗法。具体目的包括：1）生成AT1AR-T4糖酶融合蛋白，并调整这两种蛋白质之间的接头以优化AT1AR功能表达和稳定性。 2）建立用于晶体学试验的AT1AR表达和纯化的条件。 3）结晶并确定AT1AR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着核心作用，血管紧张素受体是心血管疾病的重要治疗靶点。成功完成该提案的目标将有助于开发出更具选择性和有效的心力衰竭和高血压药物。