Filamin a binds to CCR2B and regulates its internalization

• Published in: PloS one 2010-08, Vol.5 (8), p.e12212-e12212
• Main Authors: Minsaas, Laura, Planagumà, Jesús, Madziva, Michael, Krakstad, Beate F, Masià-Balagué, Míriam, Katz, Arieh A, Aragay, Anna M
• Format: Article
• Language: English
• Subjects: Acquired immune deficiency syndrome; Actin; Actins - metabolism; AIDS; Animals; Arrestins - metabolism; Baking yeast; beta-Arrestins; Biochemistry; Cell adhesion & migration; Cell Biology; Cell Biology/Cell Signaling; Cell Biology/Cytoskeleton; Cell Line, Tumor; Cell Membrane - metabolism; Cell migration; Cell Movement; Chemokine CCL2 - pharmacology; Chemokines; Cloning; Contractile Proteins - metabolism; Dopamine; Filamins; Gene expression; Genomes; Health sciences; HIV; Human immunodeficiency virus; Humans; Hybrid systems; Immunology/Leukocyte Signaling and Gene Expression; Immunoprecipitation; Infectious diseases; Inflammatory diseases; Internalization; Isoforms; Kinases; Lamellipodia; Leukocyte migration; Localization; Membrane structures; Microfilament Proteins - metabolism; Monocyte chemoattractant protein; Monocyte chemoattractant protein 1; Monocytes; Monocytes - cytology; Monocytes - drug effects; Monocytes - metabolism; Motility; Muscle proteins; Protein Binding; Protein Transport; Proteins; Pseudopodia; Receptors, CCR2 - chemistry; Receptors, CCR2 - metabolism; Ribonucleic acid; RNA; RNA-mediated interference; Saccharomyces cerevisiae; Signal transduction; siRNA; Spatial discrimination
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0012212

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.