Pons Mònica, Izquierdo Ismael, Andreu-Carbó Mireia, Garrido Georgina, Planagumà Jesús, Muriel Olivia, Del Pozo Miguel A, Geli M Isabel, Aragay Anna M
Department of Biology, Molecular Biology Institute of Barcelona (IBMB), Spanish National Research Council (CSIC), Barcelona 08028, Spain.
Department of Biomedicine, University of Bergen, Bergen, 5009 Norway.
J Cell Sci. 2017 Jan 15;130(2):490-501. doi: 10.1242/jcs.193821. Epub 2016 Dec 1.
Proper endosomal trafficking of ligand-activated G-protein-coupled receptors (GPCRs) is essential to spatiotemporally tune their physiological responses. For the monocyte chemoattractant receptor 2 (CCR2B; one of two isoforms encoded by CCR2), endocytic recycling is important to sustain monocyte migration, whereas filamin A (FLNa) is essential for CCL2-induced monocyte migration. Here, we analyze the role of FLNa in the trafficking of CCR2B along the endocytic pathway. In FLNa-knockdown cells, activated CCR2B accumulated in enlarged EEA-1-positive endosomes, which exhibited slow movement and fast fluorescence recovery, suggesting an imbalance between receptor entry and exit rates. Utilizing super-resolution microscopy, we observed that FLNa-GFP, CCR2B and β2-adrenergic receptor (β2AR) were present in actin-enriched endosomal microdomains. Depletion of FLNa decreased CCR2B association with these microdomains and concomitantly delayed CCR2B endosomal traffic, without apparently affecting the number of microdomains. Interestingly, CCR2B and β2AR signaling induced phosphorylation of FLNa at residue S2152, and this phosphorylation event was contributes to sustain receptor recycling. Thus, our data strongly suggest that CCR2B and β2AR signals to FLNa to stimulate its endocytosis and recycling to the plasma membrane.
配体激活的G蛋白偶联受体（GPCRs）的正确内体运输对于在时空上调节其生理反应至关重要。对于单核细胞趋化因子受体2（CCR2B；CCR2编码的两种异构体之一），内吞再循环对于维持单核细胞迁移很重要，而细丝蛋白A（FLNa）对于CCL2诱导的单核细胞迁移至关重要。在这里，我们分析了FLNa在内吞途径中CCR2B运输中的作用。在FLNa敲低的细胞中，活化的CCR2B积聚在扩大的EEA-1阳性内体中，这些内体表现出缓慢移动和快速荧光恢复，表明受体进入和退出速率之间存在失衡。利用超分辨率显微镜，我们观察到FLNa-GFP、CCR2B和β2肾上腺素能受体（β2AR）存在于富含肌动蛋白的内体微结构域中。FLNa的缺失减少了CCR2B与这些微结构域的结合，并随之延迟了CCR2B的内体运输，而没有明显影响微结构域的数量。有趣的是，CCR2B和β2AR信号诱导FLNa在S2152残基处磷酸化，并且这种磷酸化事件有助于维持受体再循环。因此，我们的数据强烈表明，CCR2B和β2AR向FLNa发出信号，以刺激其胞吞作用并再循环至质膜。
