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.