pH regulation of cell surface receptors

项目摘要 细胞表面受体使细胞能够检测和响应外部环境中的信号。 G蛋白偶联受体（GPCR）是人类最大的一类细胞表面受体， 和药物最常靶向的受体家族。长期以来，人们一直认为GPCR信号是 仅限于细胞表面。然而，随着最近发现GPCR， 来自内化的内体的信号传导。内体pH值被认为是决定细胞内pH值的关键因素。 内膜信号传导的持续时间：随着内体成熟，它们的内部pH从7.0下降到5.0。 这种pH变化有可能调节GPCR-药物相互作用，调节受体构象， 并优先考虑受体-效应器相互作用。类似的pH值效应由病理引起，如癌症， 哮喘和囊性纤维化具有调节细胞表面GPCR-药物相互作用的潜力。 研究pH和GPCR功能之间的关系使我们能够识别能够 对疾病相关的酸中毒敏感。pH值的影响 对GPCR信号传导的影响仍然知之甚少。本提案的目的是填补这一知识空白。 使用我们的综合计算和实验筛选平台，我们的目标是询问 每个项目年至少有五个受体的pH敏感性。在5年项目期结束时 我们预计已经从已知的受体的优先列表中评估了至少25个GPCR， 结构、药物相互作用和临床应用。在计算上，我们可以用我们的结构- 基于信息学和虚拟配体筛选软件，称为pHinder，以识别pH敏感的 残基热点、受体和配体之间的电荷网络以及pH选择性药物化合物。 在实验上，我们将证实我们的计算预测，询问GPCR-药物相互作用， 并使用我们的新型酵母pH筛选平台筛选> 2，000种药物样化合物。 我们希望我们基于结构的计算结果，在硅片和基于细胞的药物筛选， 基于酵母的pH筛选试验将在GPCR药理学中开辟新的和意想不到的途径。 具体而言，我们预计这些努力将阐明GPCR pH传感的分子基础， 鉴定由内体和病理性pH变化调节的GPCR-药物相互作用， 并促进了在中性和/或酸性pH下选择性调节受体的药物的开发。

PROJECT SUMMARY Cell surface receptors enable cells to detect and respond to signals in their external environment. G protein-coupled receptors (GPCRs) are both the largest class of cell surface receptors in humans, and the receptor family most targeted by drugs. It has long been thought that GPCR signaling was limited to the cell surface. However, this paradigm has shifted with the recent discovery of GPCR signaling from internalized endosomes. Endosomal pH is thought to be a key factor determining the duration of endomembrane signaling: as endosomes mature their internal pH declines from 7.0 to 5.0. This pH change has the potential to modulate GPCR-drug interactions, regulate receptor conformation, and prioritize receptor-effector interactions. Similar pH effects caused by pathologies such as cancer, asthma, and cystic fibrosis have the potential to modulate GPCR-drug interactions at the cell surface. Studying the relationship between pH and GPCR function enables us to identify receptors (in)capable of endomembrane signaling, and (in)sensitive to disease-related acidosis. However, the effect of pH on GPCR signaling remains poorly understood. The goal of this proposal is to fill this gap in knowledge. Using our integrated computational and experimental screening platform, we aim to interrogate the pH sensitivities of a minimum of five receptors per project year. By the end of the 5-year project period we anticipate having evaluated a minimum of 25 GPCRs from a prioritized list of receptors with known structures, drug interactions, and clinical applications. Computationally, we would use our structure- based informatics and virtual ligand screening software, known as pHinder, to identify pH-sensitive residue hot spots, charge networks between receptors and ligands, and pH-selective drug compounds. Experimentally, we would confirm our computational predictions, interrogate GPCR-drug interactions, and screen against >2,000 drug-like compounds using our novel yeast-based pH-screening platform. We expect that the outcomes of our structure-based calculations, in silico and cell-based drug screens, and yeast-based pH-screening assays will open new and unexpected avenues in GPCR pharmacology. Specifically, we anticipate that these efforts will illuminate the molecular basis GPCR pH sensing, identify GPCR-drug interactions that are (dys)regulated by endosomal and pathological pH changes, and spur the development of drugs for selectively modulating receptors at neutral and/or acidic pH.