Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits.
Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits.
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发表时间：
1993-07
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通讯作者：
J. Hepler;T. Kozasa;A. Smrcka;Melvin I. Simon;S. Rhee;P. Sternweis;A. Gilman
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作者：
J. Hepler;T. Kozasa;A. Smrcka;Melvin I. Simon;S. Rhee;P. Sternweis;A. Gilman
异源三聚体鸟嘌呤核苷酸结合蛋白（G蛋白）的Gq α亚家族成员激活磷脂酶C（PLC）。使用重组杆状病毒在昆虫（Sf 9）细胞中表达G蛋白α亚基Gq α和G11 α的互补DNA（cDNA）。只有当α亚基cDNA与编码G蛋白β和γ亚基的cDNA一起表达时，才产生活性的、非聚集的和膜相关的蛋白。重组α亚基（rGq α和rG 11 α）通过三步程序纯化，作为Sf 9细胞内源性的PLC活化α亚基。鸟苷5 '-3-（硫代）三磷酸（GTP γ S）激活rGq α和rG 11 α，表观K0.5为30 μ M;观察[35 S]GTP γ S与rGq α的结合需要类似的高浓度核苷酸。激活的rGq α和rG 11 α分别刺激纯化的PLC-β的所有三种亚型，效力等级顺序为PLC-β 1 = PLC-β 3 >或= PLC-β 2;两种α亚基也刺激PLC-β 1和PLC-β 3的程度远大于PLC-β 2（10倍）。相反，激活的rGq α和rG 11 α不能刺激PLC-δ 1或PLC-γ 1。重组Gi α 1、Gi α 2、Gi α 3、Go α（A）、Gs α和Gz α均不能刺激PLC的任何同种型。rGq α和rG 11 α对PLC-β 1的表观亲和力及其激活酶的能力与纯化脑Gq α/11 α观察到的值相似。纯化的脑β-γ亚基也刺激PLC-β的三种亚型。rGq α和rG 11 α激活PLC-β 1和PLC-β 3的能力大大超过β γ，而Gq α、G11 α和β γ与PLC-β 2大致等效;在所有情况下，α亚基比β γ更有效。对于PLC-β 1和PLC-β 2而言，α和β-γ的共同作用是非累加性的。这些结果表明，Gq α和G11 α特异性和选择性地刺激PLC的β亚型，并证实了G蛋白的Gq α亚家族的这些成员是该信号通路的生理调节剂的想法。
Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC-gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC-beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC-beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway.