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

布景主题：生物科学的健康P-Rex鸟嘌呤核苷酸交换因子（GEF）激活小GTdR的Rac后，刺激各种细胞表面受体，包括G蛋白偶联受体（GPCR）。通过它们激活Rac的能力，它们控制基因表达，细胞生长，细胞存活和运动，以及其他反应，从而调节重要的生理过程，如先天免疫，葡萄糖稳态，产热性，色素沉着和突触plasticity.We有数据表明P-Rex在GPCR贩运中的新作用。配体与GPCR的结合不仅诱导信号传导（在几秒钟内），而且通过网格蛋白介导的内吞作用（在几分钟内）使受体内化，以关闭信号传导。P-Rex缺陷促进GPCR内化，而过度表达则相反地阻断内吞作用的第一步-受体磷酸化-独立于催化Rac-GEF活性我们假设由P-Rex表达引起的GPCR细胞表面水平增加可能导致组成性细胞信号传导和应答，本项目旨在揭示P-Rex Rac-GEFs控制GPCR转运的分子机制和功能，并探索其控制P-Rex Rac-GEFs转运的潜力。雷克斯信号传导机制（18个月）：我们将通过使用图像分析和细胞分级，量化P-Rex 1和P-Rex 2对表达1-磷酸鞘氨醇（S1 PRGFP）GPCR的HEK 293细胞中受体内化的影响。我们将通过co-IP测试P-Rex或无催化活性的P-Rex* 与S1 PR-GFP、异源三聚体G蛋白和GPCR-激酶Grk 2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控制贩运所需的P-Rex残基，我们将产生贩运缺陷突变体。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Grk 2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效应。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反应延长：表达P-Rex或P-Rex* 的HEK 293 S1 PR-GFP细胞、内源性P-Rex 1敲低的PC 12 S1 PR-GFP细胞和原代鼠P-Rex无效或P-Rex 1 * 中性粒细胞。PC 12细胞和中性粒细胞表达高水平的内源性P-Rex 1，并产生已知的P-Rex 1依赖性反应，从而使我们能够确定生理重要性。它们还表达可被敏感抗体处理的内源性GPCR（例如C5 R1）。我们将测量Ca 2+通量和cAMP水平，使用P-Rex 1 * 来确定Rac依赖性，并测试通路组分的活性，例如PKA，PI 3 K，Akt，ERK，Rac 1和RhoG。我们将使用各种技术，包括成像和流式细胞术，分析细胞粘附，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）：在Vernalis实习期间，我们将开发用于筛选片段和其他小分子化合物文库的检测方法，以调节P-Rex依赖的细胞表面GPCR水平。活性化合物将作为化学探针进行评估，用作遗传操作的补充方法。

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blocks the first step of endocytosis - receptor phosphorylation - independently of catalytic Rac-GEF activity (unpublished).We hypothesise that increased GPCR cell surface levels caused by P-Rex expression may result in constitutive cell signalling and responses, and that such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.