title: Multiple signaling pathways of human interleukin-8 receptor A. Independent regulation by phosphorylation.
abstract: Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) 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 approximately 30% 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 (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ 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 PLCbeta3 phosphorylation and cross-desensitization of Ca2+ 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, Ca2+ 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 PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. richa021@mc.duke.edu
authors.forename: R M
authors.initials: RM
authors.lastname: Richardson
authors.forename: H
authors.initials: H
authors.lastname: Ali
authors.forename: B C
authors.initials: BC
authors.lastname: Pridgen
authors.forename: B
authors.initials: B
authors.lastname: Haribabu
authors.forename: R
authors.initials: R
authors.lastname: Snyderman
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D054799:Phospholipase C beta; D010766:Phosphorylation; D018123:Receptors, Interleukin / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D016209:Interleukin-8; D007527:Isoenzymes; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins