The Rac-GEF P-Rex1 Regulates Agonist-Induced GPCR Trafficking

P-Rex guanine-nucleotide exchange factors (GEFs) activate the small GTPase Rac following stimulation of a variety of cell surface receptors, including G protein-coupled receptors (GPCRs). By activating Rac, P-Rex proteins control gene expression, cell survival and motility, among other responses, and therefore play important roles in regulating physiological processes including innate immunity, glucose homeostasis, thermogenicity, pigmentation and synaptic plasticity. Ligand binding to GPCRs not only induces signalling (within seconds) but also the internalisation of the receptor by clathrin-mediated endocytosis (within minutes) to switch off signalling. Previous data from our lab for the GPCR Sphingosine-1-Phosphate Receptor 1 (S1PR1) suggested a new role of P-Rex in GPCR trafficking. P-Rex overexpression blocks the first step of agonist-induced S1PR1 endocytosis - receptor phosphorylation - independently of catalytic Rac-GEF activity (unpublished). My PhD builds on this previous research providing a comprehensive insight into the role of P-Rex1 in GPCR trafficking. Upon generating a P-Rex1 knock-out PC12-S1PR1-GFP cell line, I used imaging and cell fractionation methods to show that endogenous P-Rex1 limits the agonist-induced internalisation of S1PR1 and quantified this effect. I investigated receptor specificity, finding that the role of P-Rex1 in blocking receptor trafficking extends to all GPCRs tested, regardless of which type of heterotrimeric G protein these receptors couple to. In contrast, receptor tyrosine kinases were unaffected. Mechanistically, an early stage of receptor internalisation, receptor phosphorylation, was inhibited by P-Rex1 in a GEF-activity independent manner. I also showed that endogenous P-Rex1 levels correlate with the SDF1α-induced internalisation of CXCR4 in MDA-MB-231 and MCF7 breast cancer cell lines. Finally, I investigated the role of P-Rex1 in physiological responses of neuronal cells, revealing novel GPCR signalling-dependent roles in the activation of Rac3 and Akt, and in cell spreading, as well as constitutive roles in cAMP production, cell-cycle progression and proliferation.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2021

Author dc:creator

• Hampson, Elizabeth

Advisor dc:contributor.advisor

• Welch, Heidi

• Biology
• Cell Biology

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0635d9c9-cc51-4c26-bccc-4812b9eea85f/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.76588

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/329142