Membrane Translocation of P-Rex1 Is Mediated by G Protein βγ Subunits and Phosphoinositide 3-Kinase

Abstract

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac that is directly activated by the ␤␥ subunits of heterotrimeric G proteins and by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), which is generated by phosphoinositide 3-kinase (PI3K). G␤␥ subunits and PIP 3 are membrane-bound, whereas the intracellular localization of P-Rex1 in basal cells is cytosolic. Activation of PI3K alone is not sufficient to promote significant membrane translocation of P-Rex1. Here we investigated the subcellular localization of P-Rex1 by fractionation of Sf9 cells co-expressing P-Rex1 with G␤␥ and/or PI3K. In basal, serum-starved cells, P-Rex1 was mainly cytosolic, but 7% of the total was present in the 117,000 ؋ g membrane fraction. Co-expression of P-Rex1 with either G␤␥ or PI3K caused only an insignificant increase in P-Rex1 membrane localization, whereas G␤␥ and PI3K together synergistically caused a robust increase in membrane-localized P-Rex1 to 23% of the total. PI3K-driven P-Rex1 membrane recruitment was wortmannin-sensitive. The use of P-Rex1 mutants showed that the isolated Dbl homology/pleckstrin homology domain tandem of P-Rex1 is sufficient for synergistic G␤␥-and PI3K-driven membrane localization; that the enzymatic GEF activity of P-Rex1 is not required for membrane translocation; and that the other domains of P-Rex1 (DEP, PDZ, and IP4P) contribute to keeping the enzyme localized in the cytosol of basal cells. In vitro Rac2-GEF activity assays showed that membrane-derived purified P-Rex1 has a higher basal activity than cytosol-derived P-Rex1, but both can be further activated by PIP 3 and G␤␥ subunits.The small GTPase Rac (isoforms Rac1, Rac2, and Rac3) is a member of the Rho family of GTPases that regulates a range of important cellular functions, including gene expression, cytoskeletal structure, and reactive oxygen species formation (1). Like all small GTPases, Rac is directly activated by guaninenucleotide exchange factors (GEFs) 3 (2). The P-Rex family of Rac-GEFs is composed of P-Rex1, P-Rex2, and P-Rex2b (3-5). P-Rex1 is expressed in white blood cells and brain (3), P-Rex2 expression is more widespread but low or absent in leukocytes (4), and P-Rex2b, a splice variant of P-Rex2 that lacks the C-terminal half, is expressed mainly in the heart (5). Studies in P-Rex1-deficient mice have shown that P-Rex1 is involved in the regulation of G protein-coupled receptor (GPCR)-dependent Rac2 activation in neutrophils, production of reactive oxygen species, chemotaxis, and neutrophil recruitment to inflammatory sites (6, 7). In the neuronal PC12 cell line, RNA interference-mediated down-regulation of P-Rex1 leads to impaired neurotrophin-stimulated cell migration (8). In the endothelial HUVEC cell line, suppression of P-Rex2b levels by RNA interference results in reduced Rac1 activation and cell migration in response to sphingosine 1-phosphate (9). P-Rex family GEFs are directly and synergistically activated in vitro and in vivo by the ␤␥ subunits of heterotrimeric...

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

“…PH domains frequently interact with G␤␥ subunits (32)(33)(34)(35), and the P-Rex1 PH domain is required for G␤␥-mediated membrane recruitment (31). Importantly, alignment of the PH domains of P-Rex1 and GRK2 within the P-Rex1⅐Rac1 and GRK2⅐G␤␥ complexes positions G␤␥ into a highly negatively charged surface patch of the P-Rex1 structure with few steric clashes (Fig.…”

“…The PIP 3 and G␤␥ Binding Sites Are on the Opposite Side of P-Rex1 from the Rac1 Interface-The P-Rex1 PH domain is necessary for PIP 3 binding and coordinates P-Rex1 membrane recruitment and exchange activity (21,31). PIP 3 binding therefore plays a crucial role in P-Rex1 regulation.…”

“…In addition to PIP 3 binding, the binding of G␤␥ subunits increases P-Rex1 activity and facilitates the efficient recruitment of P-Rex1 to the plasma membrane (21,29,31). PH domains frequently interact with G␤␥ subunits (32)(33)(34)(35), and the P-Rex1 PH domain is required for G␤␥-mediated membrane recruitment (31).…”

“…For instance, P-Rex1 PH domain binding to PIP 3 is necessary for activation and membrane localization (21,31); however the mechanism by which PIP 3 binding increases P-Rex1 exchange activity is unresolved. In addition to PIP 3 binding, PH domains commonly interact with G␤␥ subunits (32)(33)(34)(35).…”

“…RTK stimulation activates phosphoinositide 3-kinase (PI3K) to phosphorylate PIP 2 to PIP 3 , whereas GPCR activation leads to dissociation of the heterotrimeric G protein into G␣ and G␤␥ subunits. Both the PIP 3 and G␤␥ subunits are thought to synergistically activate P-Rex proteins at the plasma membrane (29,31).…”