Exploring the mechanism, function and targeting potential of GPCR trafficking control by P-REX RAC-GEFs

探索 P-REX RAC-GEF 控制 GPCR 贩运的机制、功能和靶向潜力

• 批准号: 2491489
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2491489
• 关键词: Exploring; mechanism; function; targeting; potential

Theme: Bioscience for HealthP-Rex guanine-nucleotide exchange factors (GEFs) activate the small GTPase Rac upon stimulation of various cell surface receptors, including G-protein coupled receptors (GPCRs). Through their ability to activate Rac, they control gene expression, cell growth, cell survival and motility, among other responses, and thus regulate important physiological processes such as innate immunity, glucose homeostasis, thermogenicity, pigmentation and synaptic plasticity.We have data suggesting a new role of P-Rex in GPCR trafficking. Ligand binding to GPCRs not only induces signalling (within seconds) but also the internalisation of the receptor by clathrinmediated endocytosis (within minutes) to switch off signalling. P-Rex deficiency promotes GPCR internalisation, whereas overexpression, inversely, blocks the first step of endocytosis - receptor phosphorylation - independently of catalytic Rac-GEF activity (unpublished).We hypothesise that increased GPCR cell surface levels caused by P-Rex expression may result in constitutive cell signalling and responses, and that such upregulated GPCRs may be targetable.This project aims to uncover the molecular mechanism and function of GPCR trafficking control by P-Rex Rac-GEFs and explore its potential for controlling P-Rex signalling.Mechanism (18 months): We will quantify the effects of P-Rex1 and P-Rex2 on receptor internalisation in HEK293 cells which express the GPCR for sphingosine 1-phosphate (S1PRGFP), by using image analysis and cell fractionation. We will test the interactions of P-Rex or catalytically-inactive P-Rex* with S1PR-GFP, heterotrimeric G proteins and the GPCR-kinase Grk2 by co-IPs, assess which domains are required using P-Rex mutants, and test if interactionsare direct using recombinant proteins. If we can pinpoint P-Rex residues required for GPCR trafficking control, we will generate traffic-deficientmutants. We will measure if P-Rex alters GPCR ligand binding capacity, use antagonists to determine if GPCR activity is required, and test if PRex regulates Grk2 activity. To determine receptor specificity, we will measure the trafficking effects of P-Rex on different receptor classes.Function (18 months): We will elucidate if increased GPCR surface levels in P-Rex expressing cells result in prolonged signalling and responses in 3 cell types: P-Rex or P-Rex* expressing HEK293 S1PR-GFP cells, PC12 S1PR-GFP cells with knock-down of endogenous P-Rex1, and primary murine P-Rex null or P-Rex1* neutrophils. PC12 cells and neutrophils express high levels of endogenous P-Rex1 and generate responses known to be P-Rex1 dependent, thus allowing us to determine physiological importance. They also express endogenous GPCRs (e.g. C5R1) tractable by sensitive antibodies. We will measure Ca2+ fluxes and cAMP levels, using P-Rex1* to determine Rac dependence, and test activities of pathway components, e.g. PKA, PI3K, Akt, ERK, Rac1 and RhoG. We will assay cell adhesion, morphology, migration, cell cycle progression, survival and proliferation, using various techniques including imaging and flow cytometry.Targeting potential (6 months, including +3 at Vernalis): During the internship at Vernalis, we will develop assays for screening libraries of fragments and other small-molecule compounds to modulate the P-Rex dependent cell surface levels of GPCRs. Active compounds will be assessed as chemical probes for use as a complementary approach to genetic manipulation.

主题：健康生物科学P-Rex 鸟嘌呤核苷酸交换因子 (GEF) 在刺激各种细胞表面受体（包括 G 蛋白偶联受体 (GPCR)）后激活小 GTPase Rac。通过激活 Rac 的能力，它们可以控制基因表达、细胞生长、细胞存活和运动等反应，从而调节重要的生理过程，例如先天免疫、葡萄糖稳态、生热性、色素沉着和突触可塑性。我们有数据表明 P-Rex 在 GPCR 运输中的新作用。与 GPCR 结合的配体不仅诱导信号传导（几秒钟内），而且还通过网格蛋白介导的内吞作用（几分钟内）诱导受体内化以关闭信号传导。 P-Rex 缺乏促进 GPCR 内化，而过度表达则相反，阻断内吞作用的第一步 - 受体磷酸化 - 独立于催化 Rac-GEF 活性（未发表）。我们假设 P-Rex 表达引起的 GPCR 细胞表面水平增加可能导致组成型细胞信号传导和反应，并且这种上调的 GPCR 可能是可靶向的。该项目旨在 揭示 P-Rex Rac-GEF 控制 GPCR 运输的分子机制和功能，并探索其控制 P-Rex 信号传导的潜力。机制（18 个月）：我们将通过图像分析和细胞分级，量化 P-Rex1 和 P-Rex2 对表达 1-磷酸鞘氨醇 (S1PRGFP) GPCR 的 HEK293 细胞中受体内化的影响。我们将通过 co-IP 测试 P-Rex 或催化失活 P-Rex* 与 S1PR-GFP、异源三聚体 G 蛋白和 GPCR 激酶 Grk2 的相互作用，评估使用 P-Rex 突变体需要哪些结构域，并使用重组蛋白测试相互作用是否直接。如果我们能够精确定位 GPCR 运输控制所需的霸王龙残基，我们将产生运输缺陷突变体。我们将测量 P-Rex 是否改变 GPCR 配体结合能力，使用拮抗剂来确定是否需要 GPCR 活性，并测试 PRex 是否调节 Grk2 活性。为了确定受体特异性，我们将测量 P-Rex 对不同受体类别的运输影响。功能（18 个月）：我们将阐明 P-Rex 表达细胞中 GPCR 表面水平的增加是否会导致 3 种细胞类型中的信号传导和响应延长：表达 P-Rex 或 P-Rex* 的 HEK293 S1PR-GFP 细胞、敲低内源性 P-Rex1 的 PC12 S1PR-GFP 细胞、 和原代小鼠 P-Rex null 或 P-Rex1* 中性粒细胞。 PC12 细胞和中性粒细胞表达高水平的内源性 P-Rex1，并产生已知的 P-Rex1 依赖性反应，从而使我们能够确定生理重要性。它们还表达可由敏感抗体处理的内源 GPCR（例如 C5R1）。我们将测量 Ca2+ 通量和 cAMP 水平，使用 P-Rex1* 来确定 Rac 依赖性，并测试途径成分的活性，例如PKA、PI3K、Akt、ERK、Rac1 和 RhoG。我们将使用成像和流式细胞术等各种技术来测定细胞粘附、形态、迁移、细胞周期进程、存活和增殖。靶向潜力（6 个月，包括在 Vernalis 的 +3 个月）：在 Vernalis 实习期间，我们将开发用于筛选片段文库和其他小分子化合物的分析方法，以调节 GPCR 的 P-Rex 依赖性细胞表面水平。活性化合物将作为化学探针进行评估，以用作遗传操作的补充方法。