title: beta-arrestins regulate interleukin-8-induced CXCR1 internalization.
abstract: 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.
authors.affiliation: Laboratory of Molecular Immunology and Inflammation, John P. Robarts Research Institute, London, Ontario N6G 2V4, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Barlic
authors.forename: M H
authors.initials: MH
authors.lastname: Khandaker
authors.forename: E
authors.initials: E
authors.lastname: Mahon
authors.forename: J
authors.initials: J
authors.lastname: Andrews
authors.forename: M E
authors.initials: ME
authors.lastname: DeVries
authors.forename: G B
authors.initials: GB
authors.lastname: Mitchell
authors.forename: R
authors.initials: R
authors.lastname: Rahimpour
authors.forename: C M
authors.initials: CM
authors.lastname: Tan
authors.forename: S S
authors.initials: SS
authors.lastname: Ferguson
authors.forename: D J
authors.initials: DJ
authors.lastname: Kelvin
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D019390:Arrestins / Q000502:physiology*; D001692:Biological Transport; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D034284:Dynamin I; D034281:Dynamins; D011992:Endosomes / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2; D020558:GTP Phosphohydrolases / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D016209:Interleukin-8 / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics; D051381:Rats; D018121:Receptors, Cytokine / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; D015703:Antigens, CD; C000605256:Arrb1 protein, rat; D019390:Arrestins; D016209:Interleukin-8; D008164:Luminescent Proteins; D018121:Receptors, Cytokine; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; C496569:Grk2 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins