Targeted Deorphanization of G Protein-Coupled Receptors

项目摘要 G蛋白偶联受体（GPCR）包括最丰富的人膜蛋白家族， 占蛋白质编码基因组的4%。由于其众多的生理和病理作用， 大约34%的市售药物靶向GPCR。虽然GPCR是相关的可药物靶点~140 仍然是孤儿，即它们的内源性配体未被鉴定。内源性分子的鉴定， 激活受体是一个重要的步骤，有助于确定受体在生物学途径中的作用。这里我们 描述了使用基因组编辑和高通量筛选平台筛选孤儿GPCR的研究， 测试每种受体对48种不同的肽，这些肽基于其 在脑中的丰度，它们在调节分泌途径中的定位，以及序列保守性。我们有 根据其分布仔细选择了18种潜在的神经肽结合受体GPCR， 表达水平和序列保守性。我们将使用两种互补的方法进行去冗余化： 在目标1中，我们将使用CRISPR/Cas9产生GPCR敲除细胞系的功能丧失策略， 在目标2中，我们将通过产生稳定的GPCR过表达细胞来使用功能获得策略 线将使用高通量筛选测定来测量肽配体诱导的GPCR活化。 介导的变化，在这两个细胞模型的细胞内钙释放。使用这两个独立的 方法（功能丧失和功能获得），我们以前已经成功地将两种不同的孤儿 因此，我们有信心建议的研究会在一个- 这一年的获奖时间。从这一提议中获得的结果将指导未来的研究， 受体的生理作用，以及促进结构研究，寻求深入了解受体-配体 结合位点，这是开发靶向受体的新疗法的基础。

Project summary G protein-coupled receptors (GPCRs) comprise the most abundant human membrane protein family, representing 4% of the protein-coding genome. Due to their numerous physiological and pathological roles, approximately 34% of marketed drugs target a GPCR. Although GPCRs are relevant druggable targets ~140 are still orphans i.e. their endogenous ligands unidentified. The identification of the endogenous molecules that activate the receptor is an essential step that helps define the receptor’s role in biological pathways. Here we describe studies to screen orphan GPCRs using genome editing and a high-throughput screening platform, testing each receptor against 48 distinct peptides that are predicted to be neuropeptides based on their abundance in brain, their localization to the regulatory secretory pathway, and sequence conservation. We have carefully selected 18 GPCRs that are potential neuropeptide-binding receptors based on their distribution, expression levels, and sequence conservation. We will use two complementary approaches for deorphanization: in Aim 1 we will use a loss-of-function strategy by generating GPCR knockout cell lines using CRISPR/Cas9 system; and in Aim 2 we will use a gain-of-function strategy by generating stable GPCR overexpression cell lines. A high-throughput screening assay will be used to measure the peptide ligand-induced GPCR activation- mediated changes in the intracellular calcium release in these two cellular models. Using these two independent approaches (loss- and gain- of-function) we have previously successfully deorphanized two different orphan GPCRs and hence we are confident that the proposed studies will be successfully carried out within the one- year period of this award. The results obtained from this proposal will guide future studies that explore physiological roles of the receptors as well as facilitate structural studies that seek insights into receptor-ligand binding sites, which are fundamental to the development of new therapeutics targeting the receptors.