Role of lipid membrane and hydration on the oligomerization and function of PR and A2A

脂膜和水合对 PR 和 A2A 寡聚化和功能的作用

• 批准号: 8966154
• 负责人: Songi Han
• 金额: $ 33.34万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966154
• 关键词: Address; Adenosine; Adverse effects; Affinity; Biological Assay; Cell membrane; Cell surface; Characteristics; Complex; Cysteine; Data; Detection; Detergents; Development; Disease; Drug Targeting; Electron Spin Resonance Spectroscopy; Electrons; Environment; Environmental Risk Factor; FDA approved; Funding; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glass; Goals; Grant; Heart Diseases; Homo; Human; Hydration status; Knowledge; Label; Length; Ligand Binding; Light; Lipids; Liposomes; Literature; Maps; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Modeling; Molecular Sieve Chromatography; Nuclear; Pharmaceutical Preparations; Pharmacologic Substance; Physiologic pulse; Population; Property; Proteins; Protocols documentation; Proton Pump; Proxy; Reading; Research; Role; Schizophrenia; Series; Signal Transduction; Site; Site-Directed Mutagenesis; Spin Labels; Structural Protein; Structure; Surface; System; Temperature; Testing; Time; Transition Temperature; Water; Work; Yeasts; absorption; base; biophysical techniques; biophysical tools; crosslink; design; dimer; global environment; innovation; insight; method development; monomer; mutant; novel; novel strategies; protein function; protein oligomer; protein structure; public health relevance; receptor; small molecule; targeted treatment; therapeutic target; tool

 DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are an important superfamily of membrane proteins that have been a target of nearly 40% of all commercially available pharmaceuticals due to their localization at the cell surface, making them easily accessible to interact with small molecule drugs. Despite consistent funding for new drugs, only 4 of the 24 new drugs approved by the FDA in 2013 targeted the GPCR superfamily. This low percentage is likely due, in part, to a multitude of side effects that often accompany treatment, which arise in part from a lack of structural data for GPCRs and a generally poor understanding of functional consequences of GPCR oligomerization. It has become increasingly evident that GPCRs associate with each other in membranes to form (homo- or hetero-) oligomeric complexes and that these oligomers broaden the range of cell signaling. A better understanding of the factors that drive oligomerization would potentially be key for targeted therapies to, first, understand the consequence of, and then, to modulate receptor-receptor association, e.g. by designing structure-based drugs to target an oligomer population. To pursue such ambitious goals of designing and rationalizing therapeutics that target a specific GPCR oligomer, knowledge gaps in structure-dynamics-function relationships must first be targeted, requiring a number of technological and methodological innovations, as well as the identification of viable and effective GPCR models to address basic questions regarding the functional impact of oligomerization, the lipid membrane environment, and hydration. We identify two 7TM receptors, the bacterial proteorhodopsin (PR) and the full-length human adenosine A2A GPCR that serve as excellent systems to develop and test the proposed tools to determine their oligomeric state and structure in detergent and lipid membranes. Crucially, both the PR and A2A receptors have been shown to oligomerize in native lipid or cell membrane environments, making it highly significant to test key hypotheses on their structure-dynamics-function relationships. The innovation of the proposed work lies in the choice of unique biophysical tools, many of which were developed by the PI and collaborators. They include electron paramagnetic resonance methods of Gd3+ spin-based labels to sensitively capture multiple distances in the 2-6 nm regime, Overhauser dynamic nuclear polarization to directly map out membrane and protein surface hydration dynamics, and effective Yeast expression protocols for synthesizing mg quantities of A2A receptors. The combination of these unique tools permits us to cast broadly important questions, such as: (1) What is the oligomeric state of the 7TM PR and A2A in lipid membranes? (2) Do lipid membrane composition, dynamics and hydration tune oligomerization? (3) What is the functional role of oligomerization observed for PR and A2A? The emphasis of the proposed studies on elucidating 7TM oligomer structure in native lipid membrane environment, explicit comparison to structures obtained in detergent complexes and the dynamics-based approach to reading out and evaluating protein function is novel and critically important for GPCR studies.

 描述（由申请人提供）：G 蛋白偶联受体 (GPCR) 是一个重要的膜蛋白超家族，由于其定位于细胞表面，使得它们很容易被尽管对新药的资助持续不断，但 2013 年 FDA 批准的 24 种新药中只有 4 种针对 GPCR 超家族，这一比例较低可能部分归因于 GPCR 超家族。治疗常常伴随着多种副作用，部分原因是缺乏 GPCR 的结构数据以及对 GPCR 寡聚化的功能后果普遍了解不足。越来越明显的是，GPCR 在膜中相互结合形成（同源）。 - 或异质 -）寡聚复合物，并且这些寡聚体扩大了细胞信号转导的范围，更好地了解驱动寡聚化的因素可能是靶向治疗的关键，首先要了解其结果，然后进行调节。受体-受体关联，例如通过设计针对寡聚物群体的基于结构的药物，为了实现针对特定 GPCR 寡聚物的设计和合理化治疗的宏伟目标，必须首先针对结构-动力学-功能关系中的知识差距。一系列技术和方法创新，以及确定可行且有效的 GPCR 模型来解决有关寡聚化、脂质膜环境和水合作用的功能影响的基本问题，我们确定了两种 7TM 受体，即细菌。蛋白视紫红质 (PR) 和全长人腺苷 A2A GPCR 是开发和测试所提出的工具以确定其在去垢剂和脂质膜中的寡聚状态和结构的优秀系统，重要的是，PR 和 A2A 受体均已被证明可以发挥作用。寡聚体在天然脂质或细胞膜环境中，使得测试其结构-动力学-功能关系的关键假设非常重要，该工作的创新在于选择独特的生物物理工具，其中许多工具。它们包括基于 Gd3+ 自旋标记的电子顺磁共振方法，可灵敏地捕获 2-6 nm 范围内的多个距离，Overhauser 动态核极化可直接绘制膜和蛋白质表面水合动力学，以及有效的方法。用于合成毫克量 A2A 受体的酵母表达方案 这些独特工具的组合使我们能够提出广泛的重要问题，例如：(1) 7TM PR 和 7TM PR 的寡聚状态是什么。脂质膜中的 A2A？(2) 脂质膜组成、动力学和水合是否会调节寡聚化？(3) 所观察到的 PR 和 A2A 寡聚化的功能作用是什么？拟议研究的重点是阐明天然脂质膜中的 7TM 寡聚体结构。环境、与洗涤剂复合物中获得的结构的明确比较以及基于动力学的读取和评估蛋白质功能的方法对于 GPCR 研究来说是新颖且至关重要的。