Wu, Dianqing and Lee, Chang Ho and Rhee, Sue Goo and Simon, Melvin I. (1992) Activation of Phospholipase C by the α Subunits of the G_q and G_(11) Proteins in Transfected Cos-7 Cells. Journal of Biological Chemistry, 267 (3). pp. 1811-1817. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:20120330-083329571
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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.
|Additional Information:||© 1992 American Society for Biochemistry and Molecular Biology. Received for publication, September 19, 1991. This work was supported by United States Public Health Services Grant GM34236 (to M. I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Drs. David Steele, Wad Slepak, Arieh Katz, Thomas Amatruda, and John Knopf for providing some plasmids, Dr. Thomas Wilkie for critical reading of this manuscript, and Joyce Ito for help in preparing the manuscript.|
|Official Citation:||Activation of phospholipase C by the α subunits of the Gq and G11 proteins in transfected Cos-7 cells. D Q Wu, C H Lee, S G Rhee, and M I Simon J. Biol. Chem. 1992 267: 1811-7.|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||30 Mar 2012 16:19|
|Last Modified:||19 Mar 2015 23:19|
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