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（由适用提供）：G蛋白偶联受体（GPCR）是膜蛋白的重要超家族，由于它们在细胞表面上的定位，因此几乎是所有市售药物的靶标，使其易于与小分子药物相互作用。尽管为新药提供了一致的资金，但2013年FDA批准的24种新药中，只有4种针对GPCR超家族。这种低百分比可能部分归因于经常参与治疗的多种副作用，部分原因是由于缺乏GPCR的结构数据以及对GPCR寡聚功能后果的理解总体上不足。越来越多的证据表明，GPCR在形成（同型或异性）寡聚复合物中相互关联，并且这些低聚物扩大了细胞信号传导的范围。更好地理解驱动寡聚化的因素可能是目标疗法首先了解受体受体关系的结果，例如通过设计基于结构的药物以靶向低聚物种群。要追求针对特定GPCR低聚物的设计和合理化疗法的雄心勃勃的目标，必须首先针对结构动力学 - 功能关系的知识差距，需要许多技术和方法论创新，以及确定可行有效的GPCR模型，以解决有关寡聚化的基本问题，以解决寡聚化环境的基本问题。我们鉴定了两个7TM受体，分别是细菌蛋白hopopsin（PR）和全长的人腺苷A2A GPCR，它们是开发和测试所提出的工具以确定其在确定和脂质膜上确定其寡聚状态和结构的极好系统。至关重要的是，PR和A2A受体均已证明在天然脂质或细胞膜环境中会寡聚，这使得在其结构 - 动力学 - 功能关系之间测试关键假设非常重要。拟议工作的创新在于选择独特的生物物理工具，其中许多工具是由PI和合作者开发的。其中包括基于GD3+自旋标签的电子顺磁共振方法，以敏感地捕获2-6 nm政权中的多距离，过度大关系动态核极化，以直接映射膜和蛋白质表面水合动力学以及有效的酵母表达方案，用于合成A2A受体的MG量量。这些独特的工具的结合使我们能够提出广泛重要的问题，例如：（1）脂质膜中7TM PR和A2A的寡聚状态是什么？ （2）脂质膜组成，动力学和水合调子的低聚？ （3）观察到PR和A2A的低聚的功能作用是什么？在天然脂质膜环境中阐明7TM低聚物结构的拟议研究的重点，与确定复合物中获得的结构的明确比较以及基于动力的读取和评估蛋白质功能的基于动态的方法非常重要，对于GPCR研究至关重要。