Multiple Signaling Pathways of Human Interleukin-8 Receptor A

Abstract

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase C␤ (PLC␤) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by ϳ30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5-3-O-(thio)triphosphate (GTP␥S) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTP␥S binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca 2؉ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLC␤ 3 phosphorylation and cross-desensitization of Ca 2؉ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca 2؉ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLC␤ 3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways. IL-81 is a member of the structurally related family of cytokines, called chemokines, which mediate a number of biological activities including chemotaxis of leukocytes (1, 2). Cellular responses to IL-8 are initiated by specific cell surface receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins (3). Two IL-8 receptor subtypes have been identified in human neutrophils, IL-8 receptor A or CXCR1 and IL-8 receptor B or CXCR2 (4, 5). CXCR1 is specific for IL-8, whereas CXCR2 also binds other chemokines including MGSA, GRO, and NAP2, to mediate cellular responses (3, 6). Both CXCR1 and CXCR2 undergo phosphorylation and desensitizat...

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

“…8, upon IL-8 stimulation both CXCR2 and the mutant 331T induced phosphorylation of PLC␤ 3 to an extent similar to that of CXCR1 (ϳ2-fold over basal) (14). Two-dimensional phosphopeptide mapping of the PLC␤ 3 showed that CXCR1, CXCR2, and 331T mediated phosphorylation of PLC␤ 3 to the same peptides (data not shown).…”

“…The membrane was removed, and the upper surface was washed with phosphate-buffered saline and scraped, fixed, and stained. The results are represented as mean of number of cells/well (14,17). The results are representative of three separate experiments.…”

“…Stimulation of CXCR1 by IL-8 also desensitized Ca 2ϩ mobilization in response to FR, C5aR, and PAFR (12,13). Recent studies in this laboratory, using wild type and a phosphorylation-deficient mutant of CXCR1, have demonstrated that cross-regulation of CXCR1-mediated Ca 2ϩ mobilization can occur as a consequence of receptor phosphorylation or at a site distal from receptor/G protein coupling, decreasing activation of PLC (14). Whether CXCR2, like CXCR1, cross-desensitizes responses to FR or C5aR remained to be addressed.…”

“…Reactions were stopped with 1 ml of ice-cold phosphate-buffered saline containing 10 mg/ml bovine serum albumin and washed three times with the same buffer. Then cells were lysed with 0.1 N NaOH (250 l) and dried under vacuum, and bound radioactivity was counted (9,14). Nonspecific radioactivity bound was determined in the presence of 300 nM unlabeled ligand.…”

Phosphorylation of Key Serine Residues Is Required for Internalization of the Complement 5a (C5a) Anaphylatoxin Receptor via a β-Arrestin, Dynamin, and Clathrin-dependent Pathway

“…With other chemoattractant receptors, the agonist-dependent recruitment of ␤-arrestin 2 by phosphorylation-deficient mutant receptors may also occur to variable levels, depending on the receptor and the sets of residues that are mutated. This could explain why chemotaxis and the activation of MAP kinase mediated by phosphorylation-deficient mutants of chemoattractant receptors are variably affected (41)(42)(43).…”