Guanine-nucleotide exchange factors (GEFs)
GEFs (guanine-nucleotide exchange factors) activate G proteins (Fig 1) such as the small G protein Rac which regulates cell shape (Fig 2), motility, gene expression and oxygen radical formation. Rac can be activated by many different Rac-GEFs.
Figure 1. Small G proteins like Rac are active when GTP-bound and inactive when GDP-bound. GEFs activate Rac by removing bound GDP, allowing free GTP to bind. Rac-GTP transmits signals by binding to target proteins. This can elicit a multitude of cell responses, depending on the upstream signals and GEFs. Rac signals are terminated upon GTP hydrolysis through its GTPase activity, with catalysis by GAPs.
Figure 2. Phalloidin staining of the polymerised actin, showing the cytoskeletal morphology of endothelial PAE cells without (left) or with (right) active GTP-bound Rac.
P-Rex familyWe discovered the P-Rex (PIP3-dependent-Rac exchanger) family of Rac-GEFs in 2002. The P-Rex family consists of P-Rex1, P-Rex and a splice variant, P-Rex2b. They are important for leukocyte responses against bacterial and fungal infections, neuronal morphology and synaptic plasticity, and the migration of melanocytes during development. When de-regulated they contribute to inflammatory disorders, cancer growth and metastasis.
P-Rex regulationP-Rex family GEFs are coincidence detectors for the activation of GPCRs and PI3K. They are activitated by the lipid second messenger PIP3 and by the Gbg subunits of heterotrimeric G proteins PIP3 and Gbgs also localise P-Rex to the plasma membrane, where it activates Rac. P-Rex can also be activated through dephosphorylation by PP1a.
P-Rex1 in neutrophilsIn neutrophils, leukocytes which confer immunity against bacterial and fungal infections, P-Rex1 regulates GPCR-dependent Rac2 activity, adhesion, migration and ROS formation. P-Rex1 cooperates with a Rac-GEF from another family, Vav1, in controlling GPCR-dependent neutrophil responses.
Figure 3 shows in vitro Rac-GEF activity assay with full-length recombinant P-Rex1 (left, middle) or a mutant lacking ithe PH domain (right) shwoing that P-Rex1 is synergistically activated by PIP3 and Gbg, and that activation by PIP3 occurs through the PH domain. Figure 4 shows Volocity image analysis of the adhesion onto pRGD of neutrophils with (WT) or without P-Rex1/Vav1 (P1V1), stimulated with 1.5 mM fMLP for 30 min. 24 pseudocoloured images are overimposed.
P-Pex1 and P-Rex2 in the cerebellumP-Rex1 and P-Rex2 are expressed in Purkinje neurons in the cerebellum. Purkinje neurons without P-Rex2 have perturbed dendrite morphology (Fig 5), and without P-Rex1 and P-Rex2 cannot maintain synaptic plasticity (LTP). Purkinje neurons confer motor-coordination, and consequently P-Rex2 or P-Rex1/P-Rex2 deficient mice show a motor control impairment which worsens during aging.
Inhibition of P-Rex activityP-Rex proteins contributes to inflammatory conditions and to the progression and metastasis of several types of cancer. To enable us to control P-Rex activiity in the lab and potentially in disease in the future, we have recently started to develop small molecule P-Rex inhibitors. Our prototype compounds inhibit P-Rex1 and P-Rex2 in vitro and in vivo at low micomolar concentrations (Fig 6).
Figure 5. Volocity analysis of high- resolution confocal image stacks of Purkinje neurons lacking P-Rex2 (P2 Ko, such as the one on the left) or not (P2 Wt). Graphs show main dendrite width at one cell body’s distance from the nucleus (top) and main dendrite length from the nucleus to the first major branch (bottom). Mean + SE; ≤ 230 cells per condition; 2-way Anova).
Figure 6. Effect of small molecule compound #61 on the Rac-GEF activity of the isolated DH/PH domains of P-Rex1 in vitro. Mean + SE of 3 experiments (left). PDGF-stimulated spreading of PAE cells in the presence (dark grey) or absence (light grey) of eGFP-P-Rex1 upon treatment with 1 mM small molecule compound #1 for 15 min. Mean + SE of 3 experiments (right).