Regulation of human interleukin-8 receptor A: identification of a phosphorylation site involved in modulating receptor functions.
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)
Richardson
R M
RM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
DuBose
R A
RA
Ali
H
H
Tomhave
E D
ED
Haribabu
B
B
Snyderman
R
R
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018076:DNA, Complementary; D000939:Epitopes / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D018123:Receptors, Interleukin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D012694:Serine / Q000378:metabolism; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000470:Alkaloids; D015703:Antigens, CD; D018076:DNA, Complementary; D000939:Epitopes; D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D013912:Threonine; D012694:Serine; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate