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

Lead Research Organisation: University of Cambridge
Department Name: Plant Sciences

Abstract

Theme: Bioscience for Health

P-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blocks the first step of endocytosis - receptor phosphorylation - independently of catalytic Rac-GEF activity (unpublished).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 interactions
are 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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011194/1 01/10/2015 30/09/2023
1945292 Studentship BB/M011194/1 01/10/2017 30/09/2021 Elizabeth Alice Hampson
 
Title P-Rex1 Knock Out PC12 Cell Line 
Description P-Rex1 Knock Out PC12 Cell Line generated by CRISPR-Cas9 technology 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact Cell line used to address first question of project that P-Rex1 has an endogenous role is receptor trafficking. Futher cell physiology studies also performed using these cell lines. 
 
Description Exploring the targeting potential of GPCR trafficking control by P-REX RAC-GEFs 
Organisation Vernalis
Country United Kingdom 
Sector Private 
PI Contribution I have coordinated with Vernalis throughout the project so far and started a database analysis using the online CCLE for their information. I will complete an internship of ~5 months length at their labs based in Granta Park, Cambridge.
Collaborator Contribution Members of Vernalis management sit on my personal committee and guide the project/assess progress. Training for the database analysis work has been provided. Vernalis will host my internship.
Impact Still ongoing.
Start Year 2017
 
Description Sophianum School- Technasium Challenge 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Visited Sophianum School and Het College, Netherlands to introduce the students in their Technasium education stream to the Institute projects for this academic year, developing and delivering presentations and workshops based around my research and the use of animals in research as part of the Institute's commitment to the Concordat on Openness on Animals in Research. One visit in November 2018 and one visit in April 2019 delivering in total 6 full days of ~1hr sessions to a total of 200 students. Students engaged in discussion and debate around the use of animals in research, improving the animal unit, and how to create a good scientific poster. We also launched projects which we returned in April to judge. One of the projects directly related to the signalling pathway involved in this project.
Year(s) Of Engagement Activity 2018,2019
URL https://www.babraham.ac.uk/blog/2019/01/new-perspectives-on-challenge-projects-in-the-netherlands
 
Description The Cell Escape 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Instrumental in development from start of project to delivery of a pop up escape room that explains the signalling pathway I study. We have so far taken this to Cambridge Science Festival, Latitude festival, local schools and childrens clubs, particiapants from the institute, Life Lab and more reaching over 750 people. On average, the event was rated 4.8 out of 5 stars and people would be willing to pay £25 for the experience. Evokes discussions with particiapants and other members of the public about the science at Babraham Institute and the specific signalling pathway we study. Members of the public reported that the event is fun, challenging and that volunteers were lovely and engaging, even for those not especially scientifically inclined and thay they learnt a lot.
Year(s) Of Engagement Activity 2019
URL https://www.babraham.ac.uk/blog/2019/07/escape-room-project-development