Targeted Deorphanization of G Protein-Coupled Receptors

项目总结 G蛋白偶联受体(GPCRs)是人类最丰富的膜蛋白家族， 占蛋白质编码基因组的4%。由于它们具有众多的生理和病理作用， 大约34%的上市药物针对的是GPCR。虽然GPCR是相关的可用药目标~140 仍然是孤儿，即他们的内源性配体身份不明。内源分子的鉴定 激活受体是帮助确定受体在生物途径中的作用的重要步骤。在这里我们 描述使用基因组编辑和高通量筛查平台筛查孤儿GPCR的研究， 测试每个受体与48个不同的多肽的对比，这些多肽被预测为神经肽，基于它们的 大脑中的丰度，它们在调节分泌途径中的定位，以及序列保守。我们有 根据它们的分布，精心挑选了18个潜在的神经肽结合受体， 表达水平和序列保守性。我们将使用两种互补的方法来实现去孤立化： 在目标1中，我们将使用功能丧失策略，通过使用CRISPR/Cas9产生GPCR基因敲除细胞系 系统；在目标2中，我们将使用功能增益策略，通过产生稳定的gpcr过表达细胞 台词。高通量筛选试验将用于测量多肽配体诱导的GPCR激活- 在这两种细胞模型中介导细胞内钙释放的变化。使用这两个独立的 方法(功能丧失和功能获得)我们之前已经成功地使两个不同的孤儿去孤儿 因此，我们有信心建议的研究将会在以下范围内成功进行： 本奖项的年度期限。从这项提案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对受体-配体的深入了解的结构研究 结合位点，这是开发针对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.