Differential modes of regulation of CXC chemokine-induced internalization and recycling of human CXCR1 and CXCR2

Studies of human neutrophil IL-8 receptors, CXCR1 and CXCR2, have shown tha t the two receptors are differentially regulated by ELR+-CXC chemokines, th at they differ functionally and may have diverse roles in mediating the inf lammatory process. To elucidate the role of CXCR1 and CXCR2 in inflammation and to delineate the basis for the divergent regulation of these receptors by IL-8 and NAP-2, we characterized the IL-8- and NAP-2-induced mechanisms regulating the expression of each receptor, focusing on receptor internali zation and recycling. Using HEK 293 cell transfectants, IL-8 was shown to i nduce significantly higher levels of CXCR2 internalization than NAP-2, More over, although CXCR2 bound IL-8 and NAP-2 with similarly high affinity, IL- 8 functionally competed with and displaced NAP-2, and prompted high levels of internalization, similar to those induced by IL-8 alone. In a system pro viding an identical cellular milieu far reliable comparisons between CXCR1 and CXCR2, we have shown that the mechanisms controlling the internalizatio n of CXCR1 diverge from those regulating CXCR2 internalization. Whereas IL- 8-induced internalization of CXCR1 was profoundly dependent on a region of the carboxyl terminus expressing six phosphorylation sites, internalization of CXCR2 was primarily regulated by a membrane proximal domain of the carb oxyl terminus that does not express phosphorylation sites. Analysis of rece ptor re-expression on the plasma membrane indicated that at early time poin ts following removal of free ligand and incubation of the cells at 37 degre es C, receptor recycling accounted for recovery of CXCR1 and CXCR2 expressi on, whereas at later time points other processes may be involved in recepto r re-expression. Phosphorylation-independent mechanisms were shown to direc t both receptors to the recycling pathway. The differential control of CXCR 1 vs CXCR2 internalization by IL-8 and NAP-2, as well as by phosphorylation -mediated mechanisms, suggests that a chemokine and receptor-specific mode of regulation of internalization may contribute to the divergent activities of these receptors. (C) 1999 Academic Press.