Purification of P-Rex1 from neutrophils and nucleotide exchange assay.

Abstract 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)P 3 ) and by the βγ subunits of heterotrimeric G proteins (Donald et al., 2004; Rosenfeldt et al., 2004; Welch et al., 2002). Activation by PtdIns(3,4,5)P 3 occurs by means of the PH domain of P‐Rex1 and activation by Gβγ 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)P 3 –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.