Investigating G Protein Coupling of Nanodisc-solubilized Beta Adrenergic Receptor

描述（由申请人提供）：G蛋白偶联受体（GPCR）是普遍存在的信号转导物，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靶向GPCR，并且在许多情况下，这些药物产品的脱靶效应也是通过GPCR介导的。由于其固有的不稳定性和不溶性，GPCR很难研究;然而，它们在生物过程中的重要作用无疑使其成为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，通过它来溶解膜结合的蛋白质，模拟它们的天然环境，同时稳定这些分子。在纳米盘构建体内溶解GPCR需要开发能够筛选多种受体和受体亚型的配体结合的适当分析测定。硅光子器件如光学微谐振器是高度可复用的器件，其对环表面附近的折射率的微小变化敏感。生物分子可以共价连接到微环，随后的结合事件可以通过监测每个可单独寻址的微环来表征。这种方法提供了小样品体积要求的额外优点。通过将纳米盘溶解的肾上腺素能受体固定到微环谐振器平台上，我们的目标是研究这些受体对激动剂和拮抗剂的反应中G蛋白偶联和抑制素结合。还将检查受体二聚化的影响。通过结合纳米盘和微共振器技术，将开发生物测定，以表征各种配体对GPCR结合的影响，并在一个高度可复用的平台上脱靶效应。 公共卫生相关性：目前上市的药物中约有一半靶向G蛋白偶联受体（GPCR）。非常需要高度多路复用的分析平台来表征这些受体的G蛋白偶联和抑制蛋白结合，作为潜在脱靶药物作用的指标，同时将受体保持在天然样液体双层环境中。在拟议的工作中，纳米盘溶解的肾上腺素能受体将被固定在硅光学微共振器平台上，以研究受体结合对各种配体的反应。

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.