Wu D Q, Lee C H, Rhee S G, Simon M I
Division of Biology, California Institute of Technology, Pasadena 91125.
J Biol Chem. 1992 Jan 25;267(3):1811-7.
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.
采用高效瞬时转染法将对应于各种G蛋白α亚基的cDNA导入Cos-7细胞。随后用特异性G蛋白α亚基抗肽抗血清检测合成的蛋白质，并将其定位在细胞的膜部分。用[3H]肌醇预标记并转染Gαq和Gα11 cDNA的细胞在用氟化铝刺激后，[3H]肌醇磷酸的形成显著增加。与对应于磷脂酰肌醇特异性磷脂酶Cβ1（PI-PLCβ1）以及Gαq或Gα11的cDNA共转染导致肌醇磷酸形成水平更高。在Gαq和Gα11中将谷氨酰胺209残基转换为亮氨酸的突变的引入导致PI-PLC的持续激活和肌醇磷酸的高稳态水平。另一方面，用多种其他Gα亚基cDNA转染，即GαZ、GαOA、GαOB、转导素以及GαZ和GαOA的谷氨酰胺205至亮氨酸突变体，并未增加肌醇磷酸的形成。为了进一步测试G蛋白激活PI-PLC的特异性，通过使用瞬时转染细胞的洗涤膜和纯化的PI-PLCβ1制备了无细胞系统。源自Gαq和Gα11而非GαOA转染细胞的膜显示出鸟苷5'-O-硫代三磷酸（GTPγS）刺激的PIP2水解。在用源自Gα11转染细胞的膜重构的系统中观察到的活性被用特异性Gα11抗肽抗体预孵育所阻断。所有这些结果都与以下结论一致，即Gαq和Gα11 cDNA编码在存在GTPγS时特异性激活PI-PLC的蛋白质。
