RESUMEN
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)
Asunto(s)
Antígenos CD/metabolismo , Receptores de Interleucina/metabolismo , Regulación hacia Arriba , Alcaloides/farmacología , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Sitios de Unión , ADN Complementario , Epítopos/metabolismo , Humanos , Interleucina-8/farmacología , Datos de Secuencia Molecular , Mutación , Fosforilación , Pruebas de Precipitina , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-8A , Serina/metabolismo , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Treonina/metabolismo , Células Tumorales CultivadasRESUMEN
Platelet activating factor (PAF) interacts with cell surface receptors to mediate inflammatory responses. To determine the mechanisms of PAF receptor regulation, we constructed epitope-tagged human PAF receptor cDNA (ET-PAFR) and generated stable transfectants in a rat basophilic cell line (RBL-2H3 cells). The expressed receptors displayed ligand binding and functional properties similar to the native receptors in neutrophils. PAF-stimulated intracellular Ca2+ mobilization was not inhibited by pertussis toxin (PTx), whereas phosphoinositide hydrolysis and secretion were blocked by approximately 40%. The PTx-resistant secretion mediated by PAF was, however, inhibited by guanosine 5'-O-(2-thio-diphosphate) in permeabilized RBL-2H3 cells, indicating a role for PTx-insensitive G protein. In contrast to the PAF receptor, responses mediated by formylpeptide and C5a chemoattractants were inhibited by PTx. PAF stimulated a dose- and time-dependent phosphorylation of its receptor. ET-PAFR was also phosphorylated by phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP. Staurosporine caused complete inhibition of ET-PAFR phosphorylation by PMA but only partial inhibition by PAF. Receptor phosphorylation by PAF and PMA correlated with desensitization as measured by a decrease in both PAF-stimulated GTPase activity in membranes and Ca2+ mobilization in intact cells. Phosphorylation of ET-PAFR by dibutyryl cyclic AMP was not, however, associated with desensitization. These data demonstrate that a single PAF receptor population interacts with multiple G proteins to mediate its biological responses. Moreover, ET-PAFR, unlike the formylpeptide or C5a receptors, is phosphorylated by at least three kinases (most likely protein kinases A and C and a receptor kinase). The functional consequences of cellular activation by various chemoattractants may depend upon the G protein to which their receptor is coupled.