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

主题：HealthP-Rex鸟嘌呤核苷酸交换因子(GEF)的生物科学在刺激包括G蛋白偶联受体(GPCRs)在内的各种细胞表面受体时激活小GTP酶RAC。通过激活RAC，它们控制基因表达、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，如先天性免疫、葡萄糖稳态、热原性、色素沉着和突触可塑性。我们有数据表明P-Rex在GPCR运输中发挥新的作用。与GPCRs的配体结合不仅诱导信号传递(在几秒钟内)，而且通过网状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递。P-Rex缺乏促进GPCR内化，而过度表达则阻碍内吞作用的第一步--受体磷酸化--独立于催化的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构成细胞的信号和反应。本项目旨在揭示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信号转导的潜力。机制(18个月)：我们将通过图像分析和细胞分离，量化P-Rex1和P-Rex2对表达鞘氨醇1-磷酸(S1PRGFP)GPCR的HEK293细胞受体内化的影响。我们将通过共IP测试P-Rex或催化失活的P-Rex*与S1PR-GFP、异源三聚体G蛋白和GPCR-Kinase GRK2的相互作用，评估使用P-Rex突变体所需的结构域，并测试相互作用是否直接使用重组蛋白。如果我们能够准确地定位GPCR运输控制所需的P-Rex残基，我们将产生交通缺陷突变体。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。为了确定受体的特异性，我们将测量P-Rex在不同受体类别上的转运作用。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种细胞类型的信号和反应延长：表达HEK293 S1PR-GFP的P-Rex或P-Rex*细胞，内源性P-Rex1下调的PC12 S1PR-GFP细胞，以及原代小鼠P-Rex零或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，并产生已知的依赖于P-Rex1的反应，从而使我们能够确定生理重要性。它们还表达内源性GPCRs(例如C5R1)，可被敏感抗体处理。我们将测量钙离子通量和cAMP水平，使用P-REx1*来确定对RAC的依赖，并测试通路组件，如PKA、PI3K、Akt、ERK、rac1和RhoG的活性。我们将使用包括成像和流式细胞术在内的各种技术来分析细胞的黏附、形态、迁移、细胞周期进展、存活和增殖。目标潜力(6个月，包括在Vernalis的+3)：在Vernalis的实习期间，我们将开发筛选片段和其他小分子化合物文库的方法，以调节GPCRs依赖P-Rex的细胞表面水平。活性化合物将作为化学探针进行评估，作为基因操作的补充方法。

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blocks the first step of endocytosis - receptor phosphorylation - independently of catalytic Rac-GEF activity (unpublished).We hypothesise that increased GPCR cell surface levels caused by P-Rex expression may result in constitutive cell signalling and responses, and that such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