Activation of Phospholipase C by the α Subunits of the G_q and G_(11) Proteins in Transfected Cos-7 Cells

High efficiency transient transfection was used to introduce cDNA corresponding to various G protein α subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein α subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [^3H]inositol and transfected with Gα_q and Gα_(11) cDNA showed marked increases in formation of [^3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C β1 (PI-PLC β1) and to Gα_q or Gα_(11) resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in Gα_q and Gα_(11) resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other Gα subunit cDNAs, i.e. Gα_Z, Gα_(OA), Gα_(OB), transducin, and the glutamine 205 to leucine mutants of Gα_Z and of Gα_(OA) did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC β1. Membranes derived from Gα_q and Gα_(11), but not Gα(OA) transfected cells, showed guanosine 5'-O-thiotriphosphate (GTPyS)-stimulated PIP_2 hydrolysis. The activity seen in the system reconstituted with membranes derived from Gα_(11)-transfected cells was blocked by preincubation with specific Gα_(11) antipeptide antibodies. All of these results are consistent with the conclusion that Gα_q and Gα_(11) cDNA encode proteins that in the presence of GTPyS specifically activate PI-PLC