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

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blocks the first step of endocytosis - receptor phosphorylation - independently of catalytic Rac-GEF activity (unpublished).We hypothesise that increased GPCR cell surface levels caused by P-Rex expression may result in constitutive cell signalling and responses, and that such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