Filamin A Binds to CCR2B and Regulates Its Internalization

Abstract

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.

Phosphorylation of filamin A regulates chemokine receptor CCR2 recycling

“…We noticed that endosomal structures containing CCR2B appeared to be more peripherally distributed upon ligand stimulation in the human melanoma cell line M2 (lacking expression of FLNa) compared with the isogenic cell line (Minsaas et al, 2010). Initial experiments published previously had also indicated a defect in receptor recycling upon FLNa depletion (Minsaas et al, 2010). This suggests that FLNa plays roles in CCR2B endosomal trafficking, other than those previously reported.…”

“…This interaction facilitates internalization of the ligandactivated receptor into clathrin-coated pits (Minsaas et al, 2010). We noticed that endosomal structures containing CCR2B appeared to be more peripherally distributed upon ligand stimulation in the human melanoma cell line M2 (lacking expression of FLNa) compared with the isogenic cell line (Minsaas et al, 2010).…”

“…Not surprisingly, mutations in FLNa are linked to a large number of developmental diseases (Savoy and Ghosh, 2013). Among its partners are several GPCRs (Awata et al, 2001;Kim et al, 2005;Onoprishvili et al, 2003;Yu et al, 2008), including several chemokine receptors: CCR2B (one of two alternative splice forms encoded by CCR2) (Minsaas et al, 2010;Planagumà et al, 2012), CCR5 and CXCR4 (Gómez-Moutón et al, 2015;Jiménez-Baranda et al, 2007). The exact role of FLNa binding to chemokine receptors is not well understood yet, but much work indicates its function in endocytic traffic and receptor internalization (Cho et al, 2007;Kim et al, 2005;Onoprishvili et al, 2003;Seck et al, 2003).…”

“…We have previously shown that FLNa, through predominantly Ig repeats 19-23, binds to the C-terminal tail of the monocyte chemoattractant receptor 2 (CCR2, isoform B) (Minsaas et al, 2010). CCR2 and its ligand, the monocyte chemoattractant protein 1 (CCL2), are involved in a wide variety of pathophysiological inflammatory processes (Deshmane et al, 2009;Johnson et al, 2005;Sallusto and Baggiolini, 2008) and neurodegenerative disorders (Bose and Cho, 2013).…”

“…Similar to most ligand-bound GPCRs, CCL2-stimulated CCR2 is internalized in clathrin-coated vesicles (García Lopez et al, 2009), which is important to regulate its cellular signaling response (García Lopez et al, 2009;Jiménez-Sainz et al, 2003). The interaction of FLNa with the C-terminal region of CCR2B is essential for receptor internalization in endocytic vesicles and for CCL2-induced monocyte migration (Minsaas et al, 2010;Planagumà et al, 2012). Here, we show that FLNa contributes to CCR2B recycling by controlling the motility of endosomes and the entrance of the receptor into the endosomal actin microdomains that recruit cargo destined for the short-loop recycling pathway.…”