Purification of P-Rex1 from neutrophils and nucleotide exchange assay

The P-Rex family of guanine-nucleotide exchange factors (GEFs) are activators of the small GTPase Rac (Donald et al., 2004; Rosenfeldt et al., 2004; Welch et al., 2002). They are directly regulated in vitro and in vivo by the lipid second messenger phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3) and by the betagamma subunits of heterotrimeric G proteins (Donald et al., 2004; Rosenfeldt et al., 2004; Welch et al., 2002). Activation by PtdIns(3,4,5)P3 occurs by means of the PH domain of P-Rex1 and activation by Gbetagamma subunits by means of the catalytic DH domain (Hill et al., 2005). P-Rex1 and P-Rex2 also contain two DEP and two PDZ protein interaction domains, as well as homology over their COOH-terminal half to inositol polyphosphate 4-phosphatase (Donald et al., 2004; Welch et al., 2002). These domains, although not necessary for P-Rex1 activity in vitro, influence its basal and/or stimulated Rac-GEF activity, suggesting that their interaction with the DH/PH domain tandem is important for P-Rex1 function (Hill et al., 2005). P-Rex2B, a splice variant of P-Rex2, lacks the C-terminal half (Rosenfeldt et al., 2004). P-Rex1 was originally identified during a search for PtdIns(3,4,5)P3-dependent activators of Rac in neutrophils and purified to homogeneity from pig leukocyte cytosol, in which it is the major such activity (Welch et al., 2002). P-Rex1 is mainly expressed in neutrophils and regulates reactive oxygen species formation in these cells (Welch et al., 2002), whereas P-Rex2 is expressed in a wide variety of tissues but not in neutrophils (Donald et al., 2004), and P-Rex2B is expressed in the heart (Rosenfeldt et al., 2004). This Chapter describes our methods for (1) the purification of endogenous P-Rex1 from pig leukocyte cytosol, (2) the production and purification of recombinant P-Rex proteins and their substrate GTPase Rac from Sf9 cells, and (3) the in vitro assay for measuring the GEF activities of native or recombinant P-Rex proteins.