Differential Cross-regulation of the Human Chemokine Receptors CXCR1 and CXCR2

Abstract

Neutrophils and transfected RBL-2H3 cells were used to investigate the mechanism of cross-regulation of the human interleukin-8 (IL-8) receptors CXCR1 and CXCR2 by chemoattractants. In neutrophils, Ca 2؉ mobilization by the CXCR2-specific chemokine, growth-related oncogene ␣ (Gro␣), was desensitized by prior exposure to the chemoattractants N-formylated peptides (fMLP) or a complement cleavage product (C5a). In contrast, growth-related oncogene ␣ did not desensitize the latter receptors. To investigate this phenomenon, CXCR2 was stably expressed in RBL-2H3 cells and mediated phosphoinositide hydrolysis, Ca 2؉ mobilization, chemotaxis, and secretion. In cells co-expressing CXCR2 and receptors for either C5a (C5aR) or fMLP (FR), CXCR2 was cross-phosphorylated and cross-desensitized by C5a and fMLP. However, neither C5aR nor FR was cross-phosphorylated or cross-desensitized by CXCR2 activation, although CXCR1 did mediate this process. Receptor internalization induced by IL-8 was more rapid and occurred at lower doses with CXCR2 than CXCR1, although both receptors mediated equipotent chemotaxis and exocytosis in RBL. Truncation of the cytoplasmic tail of CXCR2 (331T) prolonged its signaling relative to CXCR2, increased its resistance to internalization, and induced phospholipase D activation. 331T was resistant to homologous phosphorylation and cross-phosphorylation but not cross-desensitization of its Ca 2؉ mobilization by fMLP or C5a, indicating an inhibitory site distal to receptor/G protein coupling. In contrast to CXCR2, stimulation of 331T cross-desensitized Ca 2؉ mobilization by both FR and C5aR. CXCR2 and the mutant 331T induced phospholipase C ␤ 3 phosphorylation to an extent equivalent to that of CXCR1. Taken together, these results suggest that CXCR1 and CXCR2 bind IL-8 to produce a group of equipotent responses, but their ability to generate other signals, including receptor internalization, cross-desensitization, and phospholipase D activation, are very different. The latter phenomena apparently require prolonged receptor activation, which in the case of CXCR2 is precluded by rapid receptor phosphorylation and internalization. Thus, receptors coupling to identical G proteins may trigger different cellular responses dependent on the length of their signaling time, which can be regulated by receptor phosphorylation.

Gemcitabine-induced CXCL8 expression counteracts its actions by inducing tumor neovascularization

“…CXCL8 is a potent chemotactic factor for neutrophils and has a crucial role in various types of neutrophil-mediated acute inflammation [21]. CXCR1 and CXCR2 are coordinately expressed by human neutrophils [22] and bind CXCL8 to induce a group of equipotent responses including chemotaxis and exocytosis [23]. However, they couple to distinct G proteins [24] to differentially generate other signals such as receptor internalization and phospholipase D activation [23] and these signals are required for full responsiveness of human neutrophils to CXCL8.…”

“…CXCR1 and CXCR2 are coordinately expressed by human neutrophils [22] and bind CXCL8 to induce a group of equipotent responses including chemotaxis and exocytosis [23]. However, they couple to distinct G proteins [24] to differentially generate other signals such as receptor internalization and phospholipase D activation [23] and these signals are required for full responsiveness of human neutrophils to CXCL8. Because mice do not possess the functional CXCR1 genes [25], human CXCL8 produced by gemcitabine-treated human cancer cells, could not fully induce mouse neutrophil responses and as a consequence, anti-CXCL8 antibody failed to reduce neutrophil infiltration under these conditions.…”

Evidence That Formation of Vimentin·Mitogen-activated Protein Kinase (MAPK) Complex Mediates Mast Cell Activation following FcεRI/CC Chemokine Receptor 1 Cross-talk

“…Cell Culture-Monolayer cultures of RBL-CCR1 cells (rat basophilic leukemia RBL-2H3 cells expressing human CCR1) (16) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 12% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 1 mg/ml geneticin.…”

Role of CCL7 in Type I Hypersensitivity Reactions in Murine Experimental Allergic Conjunctivitis

“…RBL-CCR1 cells and RBL-2H3 cells (ATCC CRL-2256) (38) were maintained as monolayers in DMEM (+4500 mg/l glucose, +GlutaMAX, +pyruvate) supplemented with 15% FBS and 100 U/ml penicillin and streptomycin. The cells were maintained at 3.5 3 10 5 cells per milliliter and cultured at 37˚C in 5% CO 2 .…”