beta-arrestins regulate interleukin-8-induced CXCR1 internalization

• Published in: The Journal of biological chemistry 1999-06, Vol.274 (23), p.16287-16294
• Main Authors: Barlic, J, Khandaker, M H, Mahon, E, Andrews, J, DeVries, M E, Mitchell, G B, Rahimpour, R, Tan, C M, Ferguson, S S, Kelvin, D J
• Format: Article
• Language: English
• Subjects: Animals; Antigens, CD - metabolism; Arrestins - physiology; beta-Adrenergic Receptor Kinases; beta-Arrestin 1; beta-Arrestins; Biological Transport; Cell Line; Cyclic AMP-Dependent Protein Kinases - metabolism; Dynamin I; Dynamins; Endosomes - metabolism; G-Protein-Coupled Receptor Kinase 2; Green Fluorescent Proteins; GTP Phosphohydrolases - physiology; Humans; Interleukin-8 - physiology; Luminescent Proteins - genetics; Rats; Receptors, Cytokine - metabolism; Receptors, Interleukin - metabolism; Receptors, Interleukin-8A; Recombinant Fusion Proteins - metabolism; Transferrin - metabolism; Tumor Cells, Cultured
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.23.16287

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating beta2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and beta-arrestin proteins. The importance of beta-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative beta-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, beta-arrestins, and dynamin during this process.