Research
Guanine-nucleotide exchange factors (GEFs)
Guanine-nucleotide exchange factors (GEFs) are proteins that activate small guanine-nucleotide-binding proteins (small G proteins, also known as small GTPases) of the Ras superfamily (Figure 1). Different classes of GEFs activate different families of small G proteins. GEFs are involved in a multitude of diseases. GEF mutations are known to cause several human developmental and neurological disorders, and deregulated GEF expression is associated with human cancers. GEF animal models often show impairments in the immune, nervous and cardiovascular systems.
Figure 1. Regulation of small G proteins. Small G proteins are active when GTP-bound and inactive when GDP-bound. GEFs catalyse their activation by removing bound GDP, allowing excess free cellular GTP to bind. The intrinsic GTPase activity of small G proteins catalyses auto-inactivation, but this is low unless enhanced by GAPs. Small G proteins transmit signals by binding to target proteins in their GTP-bound form. We are interested in the activators of the small G protein Rac, the Rac-GEFs.
The small G protein Rac is an essential regulator of cell shape and motility, gene expression and ROS formation. Two classes of Rac-GEFs exist, the Dbl type and the Dock type. Dbl-type Rac-GEFs include several families, which are regulated by different intracellular signals.
Figure 2. Shape of endothelial cell line PAE without (left panel) of with (right panel) active Rac. Figure 2. Shape of endothelial cell line PAE without (left panel) of with (right panel) active Rac.
The P-Rex family
We discovered a family of Rac-GEFs called P-Rex (for PtdIns(3,4,5)P3-dependent-Rac exchanger). The P-Rex family consists of P-Rex1, P-Rex2, and a splice variant, P-Rex2b. We have been studying their modes of regulation and their functional importance in several cell systems. They control leukocyte function and cerebellar Purkinje neurons, and an important role in cancer progression and metastasis is currently emerging.
Regulation of the P-Rex family P-Rex family Rac-GEFs are directly activitated by the lipid second messenger PtdIns(3,4,5)P3 and by the Gβγ subunits of heterotrimeric G proteins, which makes them ideal coincidence detectors for the activation of G protein-coupled receptors (GPCRs) and phosphoinositide 3-kinase (PI3K). P-Rex1 activation by PtdIns(3,4,5)P3 is via the PH domain (Figure 3) and activation by Gβγs via the DH domain. PtdIns(3,4,5)P3 and Gβγs also drive translocation of P-Rex1 from the cytosol to the plasma membrane, the site where it needs to be to activate Rac.
Figure 3. Regulation of P-Rex1 GEF activity. In vitro Rac-GEF activity assay with full length recombinant P-Rex1 protein (left) or with a mutated form of P-Rex1 lacking its PH domain (right), showing that the lipid second messenger PtdIns(3,4,5)P3 activates P-Rex1 via the PH domain.
Function of P-Rex1 in neutrophils P-Rex1 is strongly expressed in neutrophils, white blood cells that defend the host against bacterial and fungal infections. P-Rex1 is important for neutrophil function. It regulates GPCR-dependent Rac2 activation and ROS formation, as well as neutrophil recruitment to sites of inflammation. Recently, we have compared the neutrophil roles of P-Rex and Vav GEFs. Surprisingly, several GPCR-dependent responses that were unaffected in neutrophils lacking either the entire P-Rex family or the entire Vav family showed severe defects in cells deficient in P-Rex1 and Vav1. Hence, GEFs from different families can cooperate in GPCR signalling (Figure 4).
Figure 4. P-Rex1 and Vav1 cooperate in fMLP-stimulated neutrophil adhesion. Neutrophil adhesion onto pRGD-coated glass, stimulated with 1.5 mM fMLP for 30 min, as determined by Volocity image analysis. Neutrophils were either wild type (WT), or deficient in P-Rex1 (P1), the three Vav family members (V123), Rac2, or in combinations of P-Rex and Vav family proteins. Panels on the right show 24 overimposed and pseudocoloured images.
Function of P-Pex1 and P-Rex2 in the cerebellum P-Rex2 is strongly expressed in the brain and lung. Within the brain, P-Rex2 is mainly expressed in the Purkinje neurons of the cerebellum. P-Rex2 deficient Purkinje neurons have defects in dendrite morphology (Figure 5). Both P-Rex1 and P-Rex2 control motor-coordination, which depends on Purkinje neurons, and regulate the maintenance of synaptic plasticity (LTP) in these cells.
Figure 5. Dendrite width is reduced in Purkinje cells without P-Rex2. Volocity analysis of high resolution confocal image stacks of Purkinje neurons lacking P-Rex2 compared to control Purkinje cells. Main dendrite width was measured at a distance of one cell-body diameter from the cell body as indicated (bar graph shows mean + SE from ≤ 230 cells per condition; statistics two-way Anova).
Updated 24 August, 2011