GABA_B receptors do not internalize after baclofen treatment possibly due to lack of theβ-arrestin association ; a real-time visualization study
GABA_B receptors do not internalize after baclofen treatment possibly due to lack of theβ-arrestin association ; a real-time visualization study
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巴氯芬治疗后 GABA_B 受体不会内化，可能是由于缺乏实时可视化研究；
DOI：
10.1002/syn.21565
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Y.
中科院分区：
文献类型：
--
作者：
Sudo;Y.;Hojo;M.;Ando;Y.;Takada;M.;Murata;H.;Kurata;S.;Kanaide;M.;Nishida;N.;Uezono;Y.
激动剂诱导的GABAB受体（GABABR）内化的机制尚不清楚。为了研究这一过程，我们重点研究了GABABR与β-arrestins的相互作用，β-arrestins是大多数G蛋白偶联受体内化的关键蛋白，并使用荧光蛋白标记的GABABR和β-arrestins在真实的时间内研究了激动剂诱导的GABABR内化以及GABABR与β-arrestin 1和β-arrestin 2的相互作用。然后，我们将这些特征与μ阿片受体（μOR）的特征进行了比较，μ OR是一种研究充分的受体，与β抑制蛋白相关并共同内化。当被特异性GABABR激动剂巴氯芬刺激时，由GABAB 1aR（GB 1aR）和荧光蛋白标记的GABAB 2 R-Venus（GB 2 R-V）组成的GABABR形成功能性GABABR;它们引发G蛋白激活的内向整流钾通道以及非标记的GABABR。在共表达GB 1aR、GB 2 R-V和β-arrestin 1-Cerulean（β arr 1-C）或β-arrestin 2-Cerulean（β arr 2-C）的细胞中，真实的时间成像研究表明，巴氯芬处理既没有内化GB 2 R-V，也没有将β arr 1-C或β arr 2-C动员到细胞表面。这与G蛋白偶联受体激酶4（GRK 4）的存在无关，GRK 4与GABABR形成复合物并导致GABABR脱敏。另一方面，在共表达μOR-Venus、GRK 2和β arr 1-C或β arr 2-C的细胞中，μOR分子在细胞表面形成μOR/β arr 1或μOR/β arr 2复合物，然后以时间依赖性方式内化到细胞质中。荧光共振能量转移试验也表明，在有或没有巴氯芬刺激的情况下，GB 2 R-V和β-arrestins-C几乎不相关，而在μOR激活后，检测到μ OR-V与β-arrestins-C的强相关性。这些发现表明，GABABR未能经历激动剂诱导的内化部分是由于其未能与β抑制蛋白相互作用。Synapse 66：759-769，2012.© 2012 Wiley Periodicals，Inc.
The mechanism of agonist‐induced GABABreceptor (GABABR) internalization is not well understood. To investigate this process, we focused on the interaction of GABABR with β‐arrestins, which are key proteins in the internalization of most of the G protein‐coupled receptors, and the agonist‐induced GABABR internalization and the interaction of GABABR with β‐arrestin1 and β‐arrestin2 were investigated in real time using GABABR and β‐arrestins both of which were fluorescent protein‐tagged. We then compared these profiles with those of μ‐opioid receptors (μOR), well‐studied receptors that associate and cointernalize with β‐arrestins. When stimulated by the specific GABABR agonist baclofen, GABABR composed of GABAB1aR (GB1aR) and fluorescent protein‐tagged GABAB2R‐Venus (GB2R‐V) formed functional GABABR; they elicited G protein‐activated inwardly rectifying potassium channels as well as nontagged GABABR. In cells coexpressing GB1aR, GB2R‐V, and β‐arrestin1‐Cerulean (βarr1‐C) or β‐arrestin2‐Cerulean (βarr2‐C), real‐time imaging studies showed that baclofen treatment neither internalized GB2R‐V nor mobilized βarr1‐C or βarr2‐C to the cell surface. This happened regardless of the presence of G protein‐coupled receptor kinase 4 (GRK4), which forms a complex with GABABR and causes GABABR desensitization. On the other hand, in cells coexpressing μOR‐Venus, GRK2, and βarr1‐C or βarr2‐C, the μOR molecule formed μOR/βarr1 or μOR/βarr2 complexes on the cell surface, which were then internalized into the cytoplasm in a time‐dependent manner. Fluorescence resonance energy transfer assay also indicated scarce association of GB2R‐V and β‐arrestins‐C with or without the stimulation of baclofen, while robust association of μOR‐V with β‐arrestins‐C was detected after μOR activation. These findings suggest that GABABRs failure to undergo agonist‐induced internalization results in part from its failure to interact with β‐arrestins. Synapse 66:759–769, 2012.© 2012 Wiley Periodicals, Inc.