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The 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

基本信息

批准号：
9276861
负责人：
Songi Han
金额：
$ 20万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
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/9276861
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 Health 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 Genes Structure Surface System Temperature Testing Time Transition Temperature Water Work Yeasts absorption base biophysical techniques biophysical tools crosslink design dimer global environment innovation insight laboratory development method development monomer mutant novel novel strategies novel therapeutics protein function protein oligomer protein structure receptor small molecule targeted treatment therapeutic target tool

项目摘要

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.