Lee T W, Wise A, Cotecchia S, Milligan G
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
Biochem J. 1996 Nov 15;320 ( Pt 1)(Pt 1):79-86. doi: 10.1042/bj3200079.
Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.
对转染后表达野生型仓鼠α1B - 肾上腺素能受体或因将氨基酸残基288 - 294改变以编码人β2 - 肾上腺素能受体等效区域而产生的该受体组成型激活突变体（CAM）形式的大鼠1成纤维细胞进行了检测。表达CAMα1B - 肾上腺素能受体的细胞中肌醇磷酸生成的基础水平高于野生型受体。添加最大有效浓度的去氧肾上腺素或去甲肾上腺素后，CAMα1B - 肾上腺素能受体产生的肌醇磷酸水平显著更高，尽管该受体的稳态水平低于野生型受体。去氧肾上腺素和去甲肾上腺素刺激肌醇磷酸产生的效力在CAMα1B - 肾上腺素能受体上比在野生型受体上大约高200倍。相比之下，作用于内源性表达的内皮素ETA受体的内皮素1在两种细胞系中显示出相似的效力和最大效应。去氧肾上腺素的持续存在导致在表达野生型或CAMα1B - 肾上腺素能受体的细胞中，与磷酸肌醇酶C相连、对百日咳毒素不敏感的G蛋白G9和G11的α亚基下调。在所有激动剂浓度下，表达CAMα1B - 肾上腺素能受体的细胞中实现下调程度显著更大。然而，在该测定中，去氧肾上腺素在CAMα1B - 肾上腺素能受体上的效力仅比在野生型受体上略高。在表达野生型或CAMα1B - 肾上腺素能受体的细胞之间，G9α/G11α降解的基础速率没有可检测到的差异。在两种细胞系中，添加去氧肾上腺素均显著增加了这些G蛋白的降解速率，在CAMα1B - 肾上腺素能受体上的作用更大。激动剂在CAMα1B - 肾上腺素能受体上刺激第二信使产生以及通过刺激其降解速率来调节其相关G蛋白细胞水平的增强能力表明该形式受体与G9和G11偶联的化学计量增强。
