Role of the Cytoplasmic Tails of CXCR1 and CXCR2 in Mediating Leukocyte Migration, Activation, and Regulation

Abstract

IL-8 (or CXCL8) activates the receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB) to induce chemotaxis in leukocytes, but only CXCR1 mediates cytotoxic and cross-regulatory signals. This may be due to the rapid internalization of CXCR2. To investigate the roles of the intracellular domains in receptor regulation, wild-type, chimeric, phosphorylation-deficient, and cytoplasmic tail (C-tail) deletion mutants of both receptors were expressed in RBL-2H3 cells and studied for cellular activation, receptor phosphorylation, desensitization, and internalization. All but one chimeric receptor bound IL-8 and mediated signal transduction, chemotaxis, and exocytosis. Upon IL-8 activation, the chimeric receptors underwent receptor phosphorylation and desensitization. One was resistant to internalization, yet it mediated normal levels of β-arrestin 2 (βarr-2) translocation. The lack of internalization by this receptor may be due to its reduced association with βarr-2 and the adaptor protein-2β. The C-tail-deleted and phosphorylation-deficient receptors were resistant to receptor phosphorylation, desensitization, arrestin translocation, and internalization. They also mediated greater phosphoinositide hydrolysis and exocytosis and sustained Ca2+ mobilization, but diminished chemotaxis. These data indicate that phosphorylation of the C-tails of CXCR1 and CXCR2 are required for arrestin translocation and internalization, but are not sufficient to explain the rapid internalization of CXCR2 relative to CXCR1. The data also show that receptor internalization is not required for chemotaxis. The lack of receptor phosphorylation was correlated with greater signal transduction but diminished chemotaxis, indicating that second messenger production, not receptor internalization, negatively regulates chemotaxis.

Intracellular Domains of CXCR3 That Mediate CXCL9, CXCL10, and CXCL11 Function

“…Since we have shown previously (33) that CXCL11 is the more potent and physiological inducer of CXCR3 internalization, the tail-independent mechanism is potentially more important. This is reminiscent of the tail-and dynamin-independent process that has been described for CXCR2 internalization (52). The relative role of the internalization pathways in the in vivo function of CXCL10, CXCL9, and CXCL11 as well as the role of the CXCR3 carboxyl terminus in mediating chemotaxis is of great interest in understanding CXCR3 function in inflammatory processes.…”

Differential regulation of neutrophil chemotaxis to IL‐8 and fMLP by GM‐CSF: lack of direct effect of oestradiol

“…22 This cross-phosphorylation of chemokine receptors has been cited as a means of desensitizing them and it is generally accompanied by receptor internalization and loss from the cell surface. 23 However, the down-regulation of chemotaxis we observed was not through reduction of surface IL-8 receptors but appeared to be through a loss of signalling in response to IL-8. Interestingly, binding of a ligand of integrin b 1 to neutrophils has been shown to inhibit IL-8-mediated MAPK activation.…”

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes

“…The depression of the maximal response may be the result of insufficient time to re-establish a new equilibrium due to a slow antibody off rate. IL-8 and Gro-a stimulation of the CXCR2 receptors has been shown to result in a rapid translocation of b-arrestin, 51,61 which would support the observation of a hemi-equilibrium. However, other mechanisms cannot be completely excluded due to the limitations of the assay format.…”

“…CXCR2 rapidly internalizes in response to agonist via both b-arrestin dependent and independent mechanisms 51 through a process modulated by N-terminus, 62 ECL2 63 and cytoplasmic C-terminus structural determinants. 51,64 Antibodies that directly stimulate GPCR internalization can inhibit signaling responses through depletion of receptor from the cell surface characterized by an effect on maximal signaling responses, but not ligand potency. 65 The decrease in maximal b-arrestin recruitment observed for HY29-1 was combined with a significant shift in agonist potency.…”

“…51 Inhibition of b-arrestin recruitment to CXCR2 was assessed using the TANGO TM GPCR Assay System. This assay measures recruitment of protease-tagged arrestin protein to an activated receptor, which results in the release of a non-native transcription factor fused to the C terminus of the receptor that is then quantified using a b-lactamase reporter gene readout.…”