Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40
Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40
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人 G 蛋白偶联受体 40 的配体依赖性和非依赖性内化对抑制蛋白 3 和网格蛋白的不同要求。
DOI：
10.1016/j.cellsig.2014.07.019
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发表时间：
2014-11-01
影响因子：
4.8
通讯作者：
Zhou, Naiming
中科院分区：
文献类型：
--
作者：
Qian, Jing;Wu, Chun;Zhou, Naiming
G蛋白偶联受体40（GPR 40）被认为是增强2型糖尿病患者胰岛素分泌的一个有吸引力的靶点。已经发现GPR 40与Gq蛋白偶联，导致磷脂酶C的活化和随后的细胞内Ca 2+水平的增加。然而，调节GPR 40内化和脱敏的潜在机制仍有待阐明。在本研究中，GPR 40与增强型绿色荧光蛋白（EGFP）在其C-末端融合的构建体的构建是为了通过共聚焦显微镜直接成像GPR 40的定位和内化。在稳定转染的HEK-293细胞中，GPR 40受体经历了快速激动剂诱导的内化和组成型配体非依赖性内化。我们的数据表明，激动剂介导的GPR 40的内化被高渗蔗糖处理和siRNA介导的网格蛋白重链耗竭显著阻断。相反，组成型GPR 40内化不受高渗蔗糖或敲低网格蛋白表达的影响，但受甲基-β-环糊精（m β cD）和制霉菌素治疗的影响。此外，我们使用arrestin-3-EGFP再分布测定和siRNA介导的arrestin-3和GRK 2表达的敲低的结果显示，arrestin-3和GRK 2在激动剂介导的GPR 40内化的调节中发挥重要作用，但不参与组成性GPR 40内化的调节。此外，我们的观察表明，在激动剂激活后，内化的GPR 40受体通过Rab 4/Rab 5阳性内体快速再循环回到质膜，而组成性内化的GPR 40受体通过Rab 5阳性内体再循环回到细胞表面。由于FFA受体表现出高水平的同源性，我们的观察可能适用于该家族的其他成员。(C)2014 Elsevier Inc. All rights reserved.
G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca2+ level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knockdown of clathrin expression, but it was affected by treatment with methyl-beta-cyclodextrin (m beta cD) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family. (C) 2014 Elsevier Inc. All rights reserved.