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利用β-抑制蛋白2突变体开发BRET2筛选测定法。

Development of a BRET2 screening assay using beta-arrestin 2 mutants.

作者信息

Vrecl Milka, Jorgensen Rasmus, Pogacnik Azra, Heding Anders

机构信息

Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia.

出版信息

J Biomol Screen. 2004 Jun;9(4):322-33. doi: 10.1177/1087057104263212.

Abstract

This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET(2) signal. The beta-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET(2)-based agonist and antagonist screening assays.

摘要

本研究聚焦于增强通过生物发光共振能量转移(BRET₂)技术测量的G蛋白偶联受体(GPCR)与β-抑制蛋白2(β-arr2)之间相互作用所产生的信号。基于内化特性分类的A类(β₂-肾上腺素能受体[β₂-AR])和B类(神经激肽1型受体[NK1-R])GPCR均已得到分析。研究评估了使用(1)β-arr2磷酸化非依赖性突变体(β-arr2 R169E)和(2)与网格蛋白包被小泡成分相互作用能力缺陷的β-arr2突变体(β-arr2 R393E、R395E和β-arr2 373截短突变体)是否能增强BRET₂信号。对于B类受体,比较野生型(wt)和突变型β-arr2所获结果时,激动剂促进的BRET₂信号无显著差异。然而,对于A类受体,缺乏AP-2或同时缺乏AP-2和网格蛋白结合位点的β-arr2突变体使BRET₂信号增加了两倍多。这组数据表明,这些β-arr2突变体无法与网格蛋白包被小泡成分相互作用,可能阻止了它们从受体上快速解离,从而产生增强且更稳定的BRET₂信号。β-arr2 R393E、R395E突变体还增强了与GPCR家族其他成员(神经肽Y2型受体[NPY2-R]和TG1019受体)的信号窗口,并成功应用于基于全板BRET₂的激动剂和拮抗剂筛选试验。

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