Vines Charlotte M, Revankar Chetana M, Maestas Diane C, LaRusch Leah L, Cimino Daniel F, Kohout Trudy A, Lefkowitz Robert J, Prossnitz Eric R
Department of Cell Biology & Physiology, The University of New Mexico Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
J Biol Chem. 2003 Oct 24;278(43):41581-4. doi: 10.1074/jbc.C300291200. Epub 2003 Aug 28.
Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knockout cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonstrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the post-endocytic trafficking of GPCRs.
抑制蛋白介导大多数G蛋白偶联受体（GPCR）的磷酸化依赖性脱敏、内化及信号级联反应的起始。许多GPCR通过抑制蛋白依赖性机制进行激动剂介导的内化，其中抑制蛋白充当受体与内吞蛋白之间的衔接子。为了解抑制蛋白在N-甲酰肽受体（FPR）转运中的作用，我们在缺乏内源性抑制蛋白2和抑制蛋白3的小鼠胚胎成纤维细胞系（MEF，抑制蛋白缺陷型）中稳定表达FPR。我们将这些细胞中FPR的内化和再循环动力学与同基因野生型MEF细胞系进行比较。在缺乏抑制蛋白的情况下，FPR的内化未发生改变。由于FPR内化后仍与抑制蛋白结合，我们研究了抑制蛋白缺陷型细胞中FPR的再循环速率是否改变。虽然FPR能够在野生型细胞中再循环，但在抑制蛋白双敲除细胞中，受体再循环基本不存在。用抑制蛋白2或抑制蛋白3重建抑制蛋白缺陷型细胞系可恢复受体再循环。共聚焦荧光显微镜研究表明，在抑制蛋白缺陷型细胞中，FPR可能被困在核周再循环区室中。这些观察结果表明，虽然FPR在没有抑制蛋白的情况下可以内化，但内化的受体向细胞表面的再循环受到阻碍。我们的结果表明抑制蛋白在GPCR的内吞后转运中具有新的作用。
