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 (Pt-dIns 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 G8, G1, 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. Gβ 5γ2 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 stimu-late PtdIns 3-kinase. The β1γ 2, β1γ12, and β 113 dimers all activated PtdIns 3-kinase about 26-fold with 4-25 nM EC50 values. The β111 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 Pt-dIns 3-kinase was examined using y 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 gera nylgeranyl 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.