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器，作为药物靶点受到高度追捧。目前，有一半的上市治疗药物以gpcr为靶标，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难以研究；然而，它们在生物过程中的重要作用确定了这是一个令人信服和值得追求的目标。脂质双层纳米圆盘作为一种方便的形式出现，通过它可以溶解膜结合蛋白，模拟它们的天然环境，同时稳定这些分子。在纳米圆盘结构中溶解gpcr需要开发合适的分析方法，能够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非常敏感。生物分子可以共价地附着在微环上，随后的结合事件可以通过监测每个单独可寻址的微环来表征。这种方法提供了小样本量要求的额外优势。通过将纳米圆盘溶解的肾上腺素能受体固定在微环谐振器平台上，我们旨在研究这些受体对激动剂和拮抗剂的反应中G蛋白的偶联和抑制蛋白的结合。受体二聚化的影响也将被检查。通过纳米圆盘和微环谐振器技术的结合，生物测定将被开发出来，以在一个高度可复合的平台上表征各种配体对GPCR结合和脱靶效应的影响。

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.