Investigating G Protein Coupling of Nanodisc-solubilized Beta Adrenergic Receptor

研究纳米圆盘溶解的 β 肾上腺素受体的 G 蛋白偶联

• 批准号: 8316845
• 负责人: Courtney Sloan
• 金额: $ 4.78万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-16 至 2013-05-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8316845
• 关键词: Accounting; Adipose tissue; Adrenergic Receptor; Agonist; Annexins; Arrestins; Binding; Binding Proteins; Biological; Biological Assay; Biological Markers; Biological Models; Biological Process; Blood Vessels; Cardiac; Coupling; DNA; Detection; Detergents; Development; Devices; Dimerization; Drug Delivery Systems; Drug Receptors; Encapsulated; Endocytosis; Environment; Event; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Immobilization; Ions; Label; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Liver; Malignant Neoplasms; Marketing; Mediating; Membrane; Membrane Proteins; Methodology; Methods; Molecular; Monitor; Muscle; Nutrient; Optics; Peptides; Pharmacologic Substance; Phospholipids; Play; Post-Translational Protein Processing; Process; Protein Binding; Proteins; R7 Virus; Recombinants; Refractive Indices; Role; Sampling; Screening procedure; Semiconductors; Serine; Signal Transduction; Silicon; Solutions; Surface; Technology; Therapeutic; Tissues; Transducers; Work; base; beta-adrenergic receptor; drug development; high throughput screening; in vivo; monoamine; nanodisk; nanoscale; neurotransmission; photonics; prevent; protein expression; protein protein interaction; receptor; receptor binding; receptor function; response; sensor; therapeutic target

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) are ubiquitous signal transducers, highly sought after as pharmaceutical targets. Currently, half of all marketed therapeutics target GPCRs, and in many cases the off-target effects of these pharmaceutical products are also mediated through GPCRs. Due to their inherent instability and insolubility GPCRs are difficult to study; however, their essential role in biological processes certainly establishes this as a compelling and worthwhile pursuit. Lipid bilayer nanodiscs have emerged as a convenient format by which to solubilize membrane-bound proteins, mimicking their native environment while simultaneously stabilizing these molecules. Solubilizing GPCRs within the nanodisc construct necessitates the development of appropriate analytical assays capable of screening the ligand binding of multiple receptors and receptor subtypes. Silicon photonic devices such as optical microring resonators are highly multiplexable devices that are sensitive to small changes in refractive index near the ring surface. Biomolecules can be covalently attached to microrings and subsequent binding events can be characterized by monitoring each of the individually addressable microrings. This methodology offers the additional advantage of small sample volume requirements. By immobilizing nanodisc-solubilized ¿ adrenergic receptors onto the microring resonator platform, we aim to investigate G protein coupling and ¿ arrestin binding of these receptors in response to both agonists and antagonists. The effects of receptor dimerization will also be examined. By combining the nanodisc and microring resonator technologies, bioassays will be developed to characterize the effects of various ligands on GPCR binding and off-target effects in a highly multiplexable platform. PUBLIC HEALTH RELEVANCE: Approximately half of currently marketed pharmaceuticals target G protein coupled receptors (GPCRs). There is a significant need for highly multiplexed analysis platforms to characterize G protein coupling and arrestin binding of these receptors as indicators of potential off-target drug effects while maintaining the receptors in a native-like liid bilayer environment. In the proposed work, nanodisc-solubilized adrenergic receptors will be immobilized on silicon optical microring resonator platforms to investigate receptor binding in response to various ligands.

描述(申请人提供)：G蛋白偶联受体(GPCRs)是普遍存在的信号转导，作为药物靶标备受追捧。目前，所有上市的治疗药物中有一半是针对GPCRs的，在许多情况下，这些药物的非靶点效应也是通过GPCRs调节的。由于其固有的不稳定性和不溶性，GPCRs很难研究；然而，它们在生物过程中的基本作用肯定确立了这是一个令人信服和值得追求的目标。脂质双层纳米盘已经成为一种方便的形式，通过它可以溶解膜结合的蛋白质，模拟它们的自然环境，同时稳定这些分子。在纳米盘结构中溶解GPCRs需要开发适当的分析方法来筛选多种受体和受体亚型的配体结合。硅光子器件，如光学微环谐振器，是高度可复用的器件，对环表面附近折射率的微小变化很敏感。生物分子可以共价连接到微微球上，并且随后的结合事件可以通过监测每个单独可寻址的微微球来表征。这种方法提供了样本量要求小的额外优势。通过将纳米盘溶解的肾上腺素能受体固定在微环共振器平台上，我们的目标是研究这些受体对激动剂和拮抗剂的G蛋白偶联和arrestin结合。我们还将研究受体二聚化的影响。通过结合纳米盘和微环谐振器技术，将开发生物检测方法，以在高度可复用的平台上表征不同配体对GPCR结合和脱靶效应的影响。 公共卫生相关性：目前上市的药物中约有一半针对G蛋白偶联受体(GPCRs)。需要高度多元化的分析平台来表征这些受体的G蛋白偶联和arrestin结合，以此作为潜在的脱靶药物效应的指示物，同时将受体保持在天然的LiID双层环境中。在这项拟议的工作中，纳米盘溶解的肾上腺素能受体将被固定在硅光微环共振器平台上，以研究受体与各种配体的结合。

项目成果

Nonlinear analyte concentration gradients for one-step kinetic analysis employing optical microring resonators.

• DOI: 10.1021/ac300478f
• 发表时间: 2012-07-03
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Marty, Michael T.;Sloan, Courtney D. Kuhnline;Bailey, Ryan C.;Sligar, Stephen G.
• 通讯作者: Sligar, Stephen G.

Interfacing lipid bilayer nanodiscs and silicon photonic sensor arrays for multiplexed protein-lipid and protein-membrane protein interaction screening.

• DOI: 10.1021/ac3037359
• 发表时间: 2013-03-05
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Sloan, Courtney D. Kuhnline;Marty, Michael T.;Sligar, Stephen G.;Bailey, Ryan C.
• 通讯作者: Bailey, Ryan C.