Regulation of Human Interleukin-8 Receptor A: Identification of a Phosphorylation Site Involved in Modulating Receptor Functions

Abstract

The human type A interleukin-8 receptor (IL-8RA) was modified to express an amino-terminal epitope tag and stably overexpressed in a rat basophilic leukemia cell line (RBL-2H3). This receptor (ET-IL-8RA) displayed functional properties similar to those of the native receptor in neutrophils in that exposure to IL-8 stimulated GTPase activity, phosphoinositide (PI) hydrolysis, intracellular calcium mobilization, and degranulation in a pertussis toxin (PTx) susceptible fashion. IL-8 induced dose- and time-dependent phosphorylation of ET-IL-8RA. Phorbol 12-myristate 13-acetate (PMA) treatment also resulted in phosphorylation of the receptor although to a lesser extent. Staurosporine totally blocked PMA-induced phosphorylation but only partially inhibited IL-8-mediated phosphorylation. Phosphorylation of ET-IL-8RA correlated with its desensitization as measured by GTPase activation and calcium mobilization. To determine the role of phosphorylation in IL-8RA signal transduction, three mutants lacking specific serine and threonine residues located at the C-terminal of this receptor were constructed by site-directed mutagenesis (M1, M2, and M3). The mutated receptors expressed in RBL-2H3 cells displayed pharmacological properties (Kd approximately 2-2.8 nM and Bmax approximately 3-3.5 pmol/mg of protein) similar to those of the wild-type ET-IL-8RA. M2 and M3, but not M1, showed a marked decrease in IL-8-induced phosphorylation compared to the wild-type receptor. M2 and M3 but not M1 were resistant to PMA-mediated phosphorylation and desensitization and were also more resistant to homologous desensitization than M1 or ET-IL-8RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Human Chemokine Receptors CXCR4

“…quence (YPYDVPDYA) was inserted between the NH 2 -terminal initiator methionine and the second amino acid of human CXCR4 by polymerase chain reaction methods as described previously for other chemoattractant receptors (27,28). The same epitope tag was also placed at the COOH-terminal end before the stop codon in some constructs.…”

“…Cell Culture and Transfection-RBL-2H3 cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 g/ml) (27,28). RBL cells (1 ϫ 10 7 cells) were transfected by electroporation with pRK5 containing the receptor cDNAs (25 g) along with pcDNA3 (5 g) plasmid containing the geneticin-resistant marker and clones were isolated as described previously (27,28).…”

“…RBL cells (1 ϫ 10 7 cells) were transfected by electroporation with pRK5 containing the receptor cDNAs (25 g) along with pcDNA3 (5 g) plasmid containing the geneticin-resistant marker and clones were isolated as described previously (27,28).…”

Desensitization of N-Formylpeptide Receptor-mediated Activation Is Dependent upon Receptor Phosphorylation

“…G protein-coupled receptor kinases are implicated in the light-stimulated phosphorylation of rhodopsin (9) and the agonist-dependent phosphorylation of the ␤ 2 -adrenergic receptor (5). Recently, the FPR as well as additional chemoattractant receptors, such as the C5a and IL-8 receptors, have been shown to become rapidly phosphorylated in a ligand dose-dependent manner (10,11). Phosphorylation of the FPR was demonstrated in the human cell line HL-60 (10), which expresses endogenous FPR after terminal differentiation with agents such as dibutyryl cAMP, as well as in an FPR-transfected rat basophilic cell line, RBL-2H3 (12).…”

“…This latter form of desensitization is at least in part mediated by second messenger-activated kinases, such as protein kinase C (11). The existence of a novel intermediate form of desensitization has been suggested by observations of the desensitization of FPR-mediated inositol 1,4,5-trisphosphate generation and calcium mobilization by C5a and IL-8 in the absence of FPR phosphorylation (16).…”