Differential sensitivity of phosphatidylinositol 3-kinase p110gamma to isoforms of G protein betagamma dimers.
The ability of G protein alpha and betagamma subunits to activate the p110gamma isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110gamma form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go alpha subunits were unable to activate PtdIns 3-kinase. Dimers containing Gbeta(1-4) complexed with gamma2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gbeta5gamma2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cbeta. A series of dimers with beta subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of betagamma with phosducin (beta1H311Agamma2, beta1R314Agamma2, and beta1W332Agamma2) was tested, and only beta1W332Agamma2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the beta1 subunit complexed with a panel of different Ggamma subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The beta1gamma2, beta1gamma10, beta1gamma12, and beta1gamma13 dimers all activated PtdIns 3-kinase about 26-fold with 4-25 nm EC50 values. The beta1gamma11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110gamma form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the gamma subunit in determining the activation of PtdIns 3-kinase was examined using gamma subunits with altered CAAX boxes directing the addition of farnesyl to the gamma2 subunit and geranylgeranyl to the gamma1 and gamma11 subunits. Replacement of the geranylgeranyl group of the gamma2 subunit with farnesyl inhibited the activity of beta1gamma2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the gamma1 and gamma11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all beta subunits, with the exception of beta5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the gamma subunit and its prenyl group markedly affects the ability of the betagamma dimer to stimulate PtdIns 3-kinase.
Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia 22908-0735, USA.
Kristi R
KR
Kerchner
Robert L
RL
Clay
Gavin
G
McCleery
Nikki
N
Watson
William E
WE
McIntire
Chang-Seon
CS
Myung
James C
JC
Garrison
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015151:Immunoblotting; D007313:Insecta; D050356:Lipid Metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D014965:X-Rays
D018076:DNA, Complementary; D020033:Protein Isoforms; D019869:Phosphatidylinositol 3-Kinases; D019204:GTP-Binding Proteins; D002118:Calcium