Differential Sensitivity of P-Rex1 to Isoforms of G Protein βγ Dimers
Journal of Biological Chemistry
P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)trisphosphate and the G␤␥ subunits of heterotrimeric G proteins. We examined which of the multiple G protein ␣ and ␤␥ subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF 4 ؊ activated G s , G i , G q , G
... s , G i , G q , G 12 , or G 13 ␣ subunits were unable to activate P-Rex1. G␤␥ dimers containing G␤ 1-4 complexed with ␥ 2 stimulated P-Rex1 activity with EC 50 values ranging from 10 to 20 nM. G␤ 5 ␥ 2 was not able to stimulate P-Rex1 GEF activity. Dimers containing the ␤ 1 subunit complexed with a panel of different G␥ subunits varied in their ability to stimulate P-Rex1. The ␤ 1 ␥ 3 , ␤ 1 ␥ 7 , ␤ 1 ␥ 10 , and ␤ 1 ␥ 13HA dimers all activated P-Rex1 with EC 50 values ranging from 20 to 38 nM. Dimers composed of ␤ 1 ␥ 12 had lower EC 50 values (ϳ112 nM). The farnesylated ␥ 11 subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (␤ 1 ␥ 11 ) were also less effective at activating P-Rex1. These findings suggest that the composition of the G␤␥ dimer released by receptor activation may differentially activate P-Rex1. Heterotrimeric G proteins (G proteins), 2 monomeric G proteins (small G proteins), and the p110-␥ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) all play important roles in chemotaxis of neutrophils and other cells of hematopoietic lineage (1-4). Chemotaxis is a complex process that involves the functional coordination of a diverse array of proteins to produce cellular movement via rearrangements of the actin cytoskeleton (5). The activation of Rac, a small G protein, is a key step in chemotaxis (6). Rac is a member of the Rho subfamily of small G proteins. Like the other small G proteins, Rac is a binary switch that is inactive in the GDP bound state and active in the GTP bound state. The GDP to GTP exchange activity of Rac is regulated by at least three groups of proteins: GDP dissociation inhibitors, GTPase activating proteins, and GEFs (guanine nucleotide exchange factors). GDP dissociation inhibitors stabilize the GDP bound state of Rac, whereas GTPase activating proteins enhance the intrinsic GTPase activity of Rac. Although Rac activation can occur as a result of inhibition of GDP Downloaded from ability of a panel of G␤␥ dimers to activate P-Rex1. Moreover, as several guanine nucleotide exchange factors (p115 RhoGEF, LARG, and PDZ-RhoGEF) have been shown to be stimulated by the heterotrimeric G protein G␣ 13 (24 -27), we also explored the ability of a panel of G protein ␣ subunits to regulate P-Rex1. We found that P-Rex1 is not modulated by activated G␣ subunits but that P-Rex1 is selectively regulated by G␤␥ subunits. EXPERIMENTAL PROCEDURES Materials-The reagents used for Sf9 cell culture and purification of G protein ␣ and ␤␥ subunits have been described (28, 29) . GDP, imidazole, HEPES, Triton X-100, and cyanogen bromide-activated agarose were from Sigma. CHAPS and GTP␥S were from Roche Molecular Biochemicals; Genapol C-100 was from Calbiochem; Ni 2ϩ -nitrilotriacetic acid Superflow resin was from Qiagen; [ 35 S]GTP␥S was from PerkinElmer Life Sciences; Source 15Q anion exchange resin and the glutathione-Sepharose 4B (GST) resin were from Amersham Biosciences; Centricon 30 concentrators were from Millipore; and Ultra 30 and 100 concentrators were from Amicon. Porcine brain-extracted L-␣phosphatidylserine and L-␣-phosphatidylcholine were purchased from Avanti Polar Lipids. Bovine liver L-␣-phosphatidylinositol and PIP 3 were purchased from Sigma. All other materials were of the highest available purity.