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| ACTIVATION OF PHOSPHOLIPASE-C BY THE ALPHA-SUBUNITS OF THE GQ AND G11 PROTEINS IN TRANSFECTED COS-7 CELLS |
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| Author(s): WU DQ, LEE CH, RHEE SG, SIMON MI |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 267 Issue: 3 Pages: 1811-1817 Published: JAN 25 1992 |
| Times Cited: 282 References: 44 |
| Abstract: 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 [H-3]inositol and transfected with G-alpha(q), and G-alpha-11 cDNA showed marked increases in formation of [H-3]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. |
| Document Type: Article |
| Language: English |
Addresses:
1. CALTECH, DIV BIOL, PASADENA, CA 91125 USA
2. NHLBI, BIOCHEM LAB, BETHESDA, MD 20892 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: HA485 |
| ISSN: 0021-9258 |
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| | | | | | | | | Record from Web of Science® | | | | | | | | | |