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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., (© 2017. Published by The Company of Biologists Ltd.)

The internalization step of endocytosis has been the focus of several laboratories during the last forty years. Unlike some other budding events in the cell, many fundamental questions regarding the molecular machinery involved in the mechanism of budding itself still remain unsolved. Over the last few years the general picture of the field has quickly evolved from the originally simplistic view which postulated that clathrin polymerization is the major force driving budding at the plasma membrane. Refinement of the assays and molecular markers to measure endocytosis in animal cells has shown that other factors in addition to the clathrin coat are required and that endocytosis can also take place through clathrin-independent mechanisms. At the same time, recent introduction of genetic approaches to study endocytosis has accelerated the identification of molecules required for this process. The isolation of endocytosis mutants in budding yeast has been especially fruitful in this respect. Preliminary comparison of the results obtained in yeast and animal cells did not seem to coincide, but further progress in both systems now suggests that part of the divergence originally seen may be due to the particular experimental approaches used rather than fundamental differences in endocytic mechanisms. In this review we present a short historical overview on the advances made in yeast and animal cells regarding the study of endocytosis, underlining both emerging similarities and still interesting differences.