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首页> 外文期刊>Biochemistry >Fusion of beta 2-adrenergic receptor to G alpha s in mammalian cells: identification of a specific signal transduction species not characteristic of constitutive activation or precoupling.
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Fusion of beta 2-adrenergic receptor to G alpha s in mammalian cells: identification of a specific signal transduction species not characteristic of constitutive activation or precoupling.

机译：β2-肾上腺素能受体融合到哺乳动物细胞中的GαS：鉴定不具有本构激活或前延宁的特异性信号转导。

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The forward and antegrade interactions that comprise the agonist receptor-G protein complex were studied in Chinese hamster fibroblasts transfected to express the beta(2)-adrenergic receptor (beta(2)AR), the beta(2)AR and the alpha-subunit of its cognate G protein (G(s)), and a protein consisting of the beta(2)AR fused at its carboxy terminus with G(alpha)(s) (beta(2)AR-G(s)). Expression levels were matched at approximately 600 fmol/mg. Basal adenylyl cyclase activities were increased with the fusion receptor membranes compared to coexpressed receptor plus G(alpha)(s), and to wild-type beta(2)AR (20.5 +/- 1.8 vs 9.0 +/- 0.88 vs 8.7 +/- 0.93 pmol min(-)(1) mg(-)(1)), confirming in mammalian cells that the fusion of beta(2)AR and G(alpha)(s) results in a state not attained by expression of unfused components. However, agonist-stimulated activities were not increased proportionally, such that the stimulation over basal of the beta(2)AR-G(s) fusion protein (1. 5-fold) was less than wild-type beta(2)AR (2.1-fold). Agonist competition studies performed in the absence of guanine nucleotide exhibited high-affinity binding sites with a lower K(H) (1.75 vs 8. 47 nM) and greater %R(H) (51% vs 44%) for beta(2)AR-G(s), but GppNHp failed to convert most of these to the low-affinity state. Functional studies with the inverse agonist ICI 118551 did not show enhanced efficacy or potency with the fusion protein. Adenylyl cyclase studies with three partial agonists with diverse structures (dobutamine, ritodrine, and phenylephrine) showed no enhancement of efficacy with beta(2)AR-G(s) and a minor trend toward enhanced potency. Taken together, these results indicate that the tethering of G(alpha)(s) to the beta(2)AR causes a conformational change in the receptor that stabilizes a species "trapped" between the non-guanine nucleotide-bound state and the GTP-bound form. Functionally the receptor is not characterized by a consistent pattern of properties ascribed to other states such as constitutive activation or precoupling, but rather represents a unique state in the transition from high- to low-affinity forms.
机译：在转染的中国仓鼠成纤维细胞中研究了包含激动剂受体-G蛋白复合物的前进和促进相互作用，以表达β（2） - 肾上腺素能受体（β（2）Ar），β（2）Ar和α-亚基其同源G蛋白（G（S））和由在其羧基末端的β（2）Ar组成的蛋白质，其羧基末端与G（α）（β（2）Ar-G）融合。表达水平在约600摩尔/毫克匹配。与共混受体加g（α）和野生型β（2）Ar（20.5 +/- 1.8 vs 9.0 + / 0.88 Vs 8.7 + / - 0.93pmol min（ - ）（1）mg（ - - ）（1）），确认哺乳动物细胞，β（2）Ar和g（α）的融合导致未经用的表达未获得的状态成分。然而，激动剂刺激的活性不会比例地增加，使得对β（2）Ar-G（S）融合蛋白（1. 5倍）基底的刺激小于野生型β（2）ar（ 2.1倍）。在没有鸟嘌呤核苷酸的情况下表现出的激动剂竞争研究表现出具有较低K（H）（1.75 Vs 8.47nm）的高亲和力结合位点，更大的β（2）的β（51％Vs 44％） AR-G（S），但GPPNHP未能将大部分转换为低亲和力状态。具有反向激动剂ICI 118551的功能性研究未显示出增强的疗效或融合蛋白效力。腺苷素环酰基酶研究与三种局部激动剂具有多种结构（多巴酚丁胺，Ritodrine和苯妥），没有提高β（2）ar-g的功效和较小的趋势趋向于增强效力。总之，这些结果表明，G（α）与β（2）Ar的束缚导致受体的构象变化，其稳定非鸟嘌呤核苷酸结合状态和GTP之间的“被困”的物种 - 行为。在功能上，受体的特征在于归因于诸如组成型激活或预延线的其他状态的一致性模式，而是表示从高到低亲和力形成的过渡中的独特状态。

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