β-Arrestins Regulate Interleukin-8-induced CXCR1 Internalization*
- Jana Barlic‡§,
- Masud H. Khandaker‡§,
- Elizabeth Mahon‡,
- Joseph Andrews‡,
- Mark E. DeVries‡§,
- Gordon B. Mitchell‡,
- Rahbar Rahimpour‡,
- Christopher M. Tan‡¶,
- Stephen S. G. Ferguson¶ and
- David J. Kelvin‡§‖
- From the ‡Laboratory of Molecular Immunology and Inflammation, John P. Robarts Research Institute, London, Ontario N6G 2V4, Canada, the §Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario N6A 5C1 Canada, and the ¶Department of Physiology, Pharmacology and Toxicology, The University of Western Ontario, and John P. Robarts Research Institute, London, Ontario N6A 5K8, Canada
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 β2-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 β-arrestin proteins. The importance of β-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative β-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, β-arrestins, and dynamin during this process.
Footnotes
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↵* This work was supported by grants from the Medical Research Council of Canada, the Medical Research Council-Juvenile Diabetes Foundation International, and the Heart and Stroke Foundation of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‖ To whom correspondence should be addressed. Tel.: 519-663-3877; Fax: 519-663-3847; E-mail: kelvin{at}rri.on.ca.
- Abbreviations:
- IL-8
-
interleukin-8
- GPCR
-
guanine nucleotide-binding protein-coupled receptor
- β2AR
-
β2-adrenergic receptor
- GRK
-
G protein-coupled receptor kinases
- GFP
-
green fluorescent protein
- HEK
-
human embryonic kidney
- RBL
-
rat basophilic leukemia
- CCV
-
clathrin-coated vesicle
- EMEM
-
Eagle’s minimal essential medium
- PBS
-
phosphate-buffered saline
-
- Received November 18, 1998.
- Revision received March 9, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
