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首页> 外文期刊>Journal of Biotechnology >Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system
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Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

机译:杆状病毒共展示系统对G蛋白偶联受体介导的腺苷酸环化酶激活的功能重建

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Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galphads), G-protein betad1gammad2 subunit dimer (Gbetad1gammad2), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galphads, Gbetad1gammad2, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galphads-coupled receptors together with Galphads, Gbetad1gammad2, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galphads and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.
机译:近来,已有证据支持功能性膜蛋白及其复合物在细胞外杆状病毒颗粒(预算病毒,BV)上的异源表达。在这项研究中,我们试图将该BV显示系统应用于检测G蛋白偶联受体(GPCR)信号。我们用四种分别编码多巴胺D1受体(DR-D1),G蛋白α-亚基(Galphads),G蛋白betad1gammad2亚基二聚体(Gbetad1gammad2)和腺苷酸环化酶VI型(ACVI)的四种重组杆状病毒的组合感染了Sf9细胞)。响应多巴胺刺激,回收的BV馏分产生cAMP。除受体和ACVI外,所有三个G蛋白亚基的共表达可导致最大反应。 BV共表达DR-D1,Galphads,Gbetad1gammad2和ACVI也对多巴胺激动剂和拮抗剂反应。此外,BV与Galphads,Gbetad1gammad2和ACVI一起表达另外两个与Galphads偶联的受体,它们的特定配体也产生了cAMP。这些结果表明受体,Galphads和ACVI的功能偶联在BV上重建。由于BV基本不含内源性GPCR,因此该BV共展示系统应被证明对开发GPCR功能测定系统非常有用。

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