Membrane translocation of P-Rex1 is mediated by G protein betagamma subunits and phosphoinositide 3-kinase.
P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac that is directly activated by the betagamma 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). Gbetagamma 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 Gbetagamma and/or PI3K. In basal, serum-starved cells, P-Rex1 was mainly cytosolic, but 7% of the total was present in the 117,000 x g membrane fraction. Co-expression of P-Rex1 with either Gbetagamma or PI3K caused only an insignificant increase in P-Rex1 membrane localization, whereas Gbetagamma 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 Gbetagamma- 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 Gbetagamma subunits.
Barber
Mark A
MA
Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom.
Donald
Sarah
S
Thelen
Sylvia
S
Anderson
Karen E
KE
Thelen
Marcus
M
Welch
Heidi C E
HC
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020662:Guanine Nucleotide Exchange Factors; C453280:PREX1 protein, human; D019869:Phosphatidylinositol 3-Kinases; D000077191:Wortmannin