beta-Arrestins regulate interleukin-8-induced CXCR1 internalization

The functional role of neutrophils during acute inflammatory responses is r egulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) tha t are rapidly desensitized and internalized upon binding their cognate chem okine ligands. The efficient re-expression of CXCR1 on the surface of neutr ophils following agonist-induced internalization suggests that CXCR1 surfac e receptor turnover may involve regulatory pathways and intracellular facto rs similar to those regulating beta(2)-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligan d-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two d ifferent cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimula tion promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalizatio n in HEK 293 cells required co-expression of G protein-coupled receptor kin ase 2 and beta-arrestin proteins. The importance of beta-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative beta-a rrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalizatio n in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Ago nist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin c oated vesicles. Our data provides a direct Link between agonist-induced int ernalization of CXCR1 and a requirement for G protein-coupled receptor kina se 2, beta-arrestins, and dynamin during this process.