Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.
High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 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 beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 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 G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha 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 beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.
Wu
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Division of Biology, California Institute of Technology, Pasadena 91125.
Lee
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Rhee
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Simon
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D000906:Antibodies; D007295:Inositol Phosphates; D007527:Isoenzymes; D046911:Macromolecular Substances; D010455:Peptides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase