Regulation of human interleukin-8 receptor A: identification of a phosphorylation site involved in modulating receptor functions.

Published

Journal Article

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)

Full Text

Duke Authors

Cited Authors

  • Richardson, RM; DuBose, RA; Ali, H; Tomhave, ED; Haribabu, B; Snyderman, R

Published Date

  • October 31, 1995

Published In

Volume / Issue

  • 34 / 43

Start / End Page

  • 14193 - 14201

PubMed ID

  • 7578017

Pubmed Central ID

  • 7578017

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00043a025

Language

  • eng

Conference Location

  • United States