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 expre ss an amino-terminal epitope tag and stably overexpressed in a rat bas ophilic leukemia cell line (RBL-2H3). This receptor (ET-IL-8RA) displa yed functional properties similar to those of the native receptor in n eutrophils in that exposure to IL-8 stimulated GTPase activity, phosph oinositide (PI) hydrolysis, intracellular calcium mobilization, and de granulation in a pertussis toxin (PTx) susceptible fashion. IL-8 induc ed dose- and time-dependent phosphorylation of ET-IL-8RA. Phorbol 12-m yristate 13-acetate (PMA) treatment also resulted in phosphorylation o f the receptor although to a lesser extent. Staurosporine totally bloc ked PMA-induced phosphorylation but only partially inhibited IL-8-medi ated phosphorylation. Phosphorylation of ET-IL-8RA correlated with its desensitization as measured by GTPase activation and calcium mobiliza tion. To determine the role of phosphorylation in IL-8RA signal transd uction, three mutants lacking specific serine and threonine residues l ocated at the C-terminal of this receptor were constructed by site-dir ected mutagenesis (M1, M2, and M3). The mutated receptors expressed in RBL-2H3 cells displayed pharmacological properties (K-d similar to 2- 2.8 nM and B(max)similar to 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 de crease in IL-8-induced phosphorylation compared to the wild-type recep tor. M2 and M3 but not M1 were resistant to PMA-mediated phosphorylati on and desensitization and were also more resistant to homologous dese nsitization than M1 or ET-IL-8RA. Following exposure to IL-8, M1 and M 3 stimulated PI hydrolysis and secretion to the same extent as wild-ty pe IL-8RA. M2, however, showed an similar to 4- and similar to 12-fold increase in IL-8-induced PI hydrolysis and secretion, respectively. T hese data suggest that the IL-8RA is susceptible to phosphorylation an d desensitization by a receptor kinase (GRK) and protein kinase C (PKC ), respectively. Moreover, the residues modified in M2 and M3 are requ ired for PKC-mediated phosphorylation and desensitization. Interesting ly, the M2 cluster appears to participate in the downregulation of IL- 8RA-mediated responses.