HORMONAL-REGULATION OF GI2 ALPHA-SUBUNIT PHOSPHORYLATION IN INTACT HEPATOCYTES
HORMONAL-REGULATION OF GI2 ALPHA-SUBUNIT PHOSPHORYLATION IN INTACT HEPATOCYTES
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DOI：
10.1042/bj2680449
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发表时间：
1990-06-01
影响因子：
4.1
通讯作者：
HOUSLAY, MD
中科院分区：
文献类型：
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作者：
BUSHFIELD, M;MURPHY, GJ;HOUSLAY, MD
肝细胞含有鸟嘌呤核苷酸结合蛋白（G蛋白）“Gi-family”的Gi 2和Gi 3形式，但不含Gi 1。抗肽抗血清AS 7和13 B显示选择性免疫沉淀Gi 2和Gi 3，抗血清CS 1免疫沉淀刺激性G蛋白Gs。用胰高血糖素、TH-胰高血糖素（[1-N-α-β-D-胰高血糖素]）之一处理完整的32 P-标记的肝细胞三硝基苯基组氨酸，12-高精氨酸]胰高血糖素）、Arg-加压素、血管紧张素-II、佛波酯TPA（12-O-十四酰基佛波13-乙酸酯）和8-溴-环AMP引起α-胰高血糖素标记的时间和剂量依赖性增加。免疫沉淀的Gi 2的亚基，其抑制低浓度的不可水解的GTP类似物鸟苷5“-[β]的能力的丧失。伽马亚氨基]三磷酸（p[NH]ppG）以抑制毛喉素刺激的腺苷酸环化酶活性（“Gi”-功能）。磷酸化的Gi-2 α的免疫沉淀在免疫沉淀试验中，通过包含转导素的C-末端十肽，而不是Gz（一种“Gi样”G-蛋白，其缺乏C-末端半胱氨酸基团，在Gi家族的其他成员中，该基团被百日咳毒素ADP-核糖基化）的C-末端十肽，以剂量依赖性方式阻断了抗血清AS 7亚基对IL-10的诱导。没有标记α-观察到Gi 3或Gs的亚基。α- Gi 2在基础状态下被标记，并且在不存在配体添加的情况下，这在15分钟内没有变化。与用加压素、血管紧张素和TPA观察到的双相剂量-效应曲线相反，胰高血糖素介导的α-羟脯氨酸标记的增加的剂量-效应曲线为Gi 2是明显的双相的，其中Gi功能的丧失导致α-Gly 2标记的高亲和力组分。由胰高血糖素引起的Gi 2。TPA、TH-胰高血糖素、血管紧张素-II和加压素在α-胰高血糖素的标记中实现了类似的最大增加。Gi 2，这大约是用高胰高血糖素浓度（1 μ M）或8-溴环AMP处理肝细胞后发现的一半。免疫沉淀的α-磷酸化氨基酸含量的分析Gi 2显示仅存在磷酸丝氨酸。肝细胞膜与[γ- 32 P]ATP和纯化的蛋白激酶C，而不是蛋白激酶A，导致标记掺入免疫沉淀的α- Gi2.如果从已经接受过显示引起α-磷酸化的配体的预先处理的细胞获得膜，则这种标记被取消。完整细胞中的Gi 2。我们认为有两个可能的位点用于磷酸化α- Gi 2;一个用于C-激酶，另一个用于未鉴定的激酶，其作用由A-激酶激活触发。
Hepatocytes contain the Gi2 and Gi3 forms of the ''Gi-family'' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and 13B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-.alpha.-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the .alpha.-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5''-[.beta..gamma.-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity (''Gi''-function). The immunoprecipitation of phosphorylated Gi-2.alpha.-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a''Gi-like'' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the .alpha.-subunits of either Gi3 or Gs was observed. .alpha.-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of .alpha.-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of .alpha.-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of .alpha.-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 .mu.M) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated .alpha.-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [.gamma.-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated .alpha.-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of .alpha.-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of .alpha.-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.