RGS2 interacts with Gs and adenylyl cyclase in living cells
RGS2 interacts with Gs and adenylyl cyclase in living cells
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DOI：
10.1016/j.cellsig.2005.05.004
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发表时间：
2006-03-01
影响因子：
4.8
通讯作者：
Chidiac, P
中科院分区：
文献类型：
--
作者：
Roy, AA;Baragli, A;Chidiac, P
G蛋白信号传导调节因子（Regulator of G Protein Signaling，RGS）蛋白阻碍异源三聚体G蛋白信号传导。RGS 2减少cAMP的产生，并似乎与腺苷酸环化酶（AC）及其刺激性G蛋白Gs相互作用。我们先前表明，绿色荧光蛋白标记的RGS 2（GFP-RGS 2）定位于HEK 293细胞的细胞核，并在与Gs α或Gs偶联的β（2）-肾上腺素能受体（β（2）AR）共表达时被募集到质膜。在这里，使用共聚焦显微镜，我们表明，各种AC亚型（ACI，ACII，ACV，ACVI）的共表达也导致GFP-RGS 2招募到质膜。生物发光共振能量转移（BRET）还用于检查RGS 2与Gs信号通路组分之间的物理相互作用。在HEK 293细胞中共表达的RGS 2-海肾荧光素酶（能量供体）和Gsa-GFP（能量受体）的融合构建体之间检测到BRET信号。在GFP-RGS 2和ACII或ACV 1与海肾荧光素酶融合之间也观察到BRET。此外，RGS 2被发现与PAR相互作用。纯化的RGS 2在GST下拉试验中选择性地结合至β（2）AR的第三胞内环，并且当GFP-RGS 2和与海肾荧光素酶融合的β（2）AR与ACIV或ACVI共表达时，在这两种蛋白质之间观察到BRET信号。在不存在共表达AC的情况下，这种相互作用低于检测限，表明效应酶稳定或促进了细胞内受体与RGS蛋白之间的结合。总之，这些结果表明RGS 2可能与受体-G蛋白效应信号复合物结合以调节GS依赖性cAMP产生。(c)2005年爱思唯尔公司All rights reserved.
Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Proteintagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gs alpha, or the Gs-coupled beta(2)-adrenergic receptor (beta(2)AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gs alpha-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACV1 fused to Renilla luciferase. Additionally, RGS2 was found to interact with the PAR. Purified RGS2 selectively bound to the third intracellular loop of the beta(2)AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and beta(2)AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production. (c) 2005 Elsevier Inc. All rights reserved.