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

• Published in: Biochemistry (Easton) 1995-10, Vol.34 (43), p.14193-14201
• Main Authors: Richardson, Ricardo M, DuBose, Robert A, Ali, Hydar, Tomhave, Eric D, Haribabu, Bodduluri, Snyderman, Ralph
• Format: Article
• Language: English
• Subjects: Alkaloids - pharmacology; Amino Acid Sequence; Animals; Antigens, CD - chemistry; Antigens, CD - genetics; Antigens, CD - metabolism; Binding Sites; DNA, Complementary; Epitopes - metabolism; Humans; Interleukin-8 - pharmacology; Molecular Sequence Data; Mutation; Phosphorylation; Precipitin Tests; Protein Kinase C - antagonists & inhibitors; Rats; Receptors, Interleukin - chemistry; Receptors, Interleukin - genetics; Receptors, Interleukin - metabolism; Receptors, Interleukin-8A; Serine - metabolism; Staurosporine; Tetradecanoylphorbol Acetate - pharmacology; Threonine - metabolism; Tumor Cells, Cultured; Up-Regulation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00043a025

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.