Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Abstract The ability of G protein α and βγ subunits to activate the p110γ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110γ form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go α subunits were unable to activate PtdIns 3-kinase. Dimers containing Gβ1–4 complexed with γ2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gβ5γ2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cβ. A series of dimers with β subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of βγ with phosducin (β1H311Aγ2, β1R314Aγ2, and β1W332Aγ2) was tested, and only β1W332Aγ2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the β1 subunit complexed with a panel of different Gγ subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The β1γ2, β1γ10, β1γ12, and β1γ13 dimers all activated PtdIns 3-kinase about 26-fold with 4–25 nm EC50 values. The β1γ11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110γ form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the γ subunit in determining the activation of PtdIns 3-kinase was examined using γ subunits with altered CAAX boxes directing the addition of farnesyl to the γ2 subunit and geranylgeranyl to the γ1 and γ11 subunits. Replacement of the geranylgeranyl group of the γ2 subunit with farnesyl inhibited the activity of β1γ2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the γ1 and γ11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all β subunits, with the exception of β5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the γ subunit and its prenyl group markedly affects the ability of the βγ dimer to stimulate PtdIns 3-kinase.