Abstract
Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t((1/2)) approximately 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-beta and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nM) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t((1/2)) < 5 s) binding of beta-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.
Highlights
FEBRUARY 16, 2007 VOLUME 282 NUMBER 7 fulfill important roles in host defense and inflammation
Characterization of Anti-pC5aR1⁄7Ser-334 mAb 32-G1—To generate phosphosite-specific antibodies that react with activated/phosphorylated C5a receptor (C5aR), mice were immunized with a synthetic C5aR C-terminal phosphopeptide that contained phosphoserine instead of serine at position 334 and at the neighboring phosphorylation sites Ser-332 and Ser-338
The mAb 32-G1 was chosen among several hybridoma clones that were derived from the fusion of splenocytes with myeloma cells and selectively reacted with the C5aR phosphopeptide but not with a non-phosphorylated version of the same peptide
Summary
FEBRUARY 16, 2007 VOLUME 282 NUMBER 7 fulfill important roles in host defense and inflammation. We investigate using site-specific phosphopeptide antibodies the kinetics of C5a-induced C5aR1⁄7Ser334 phosphorylation in cells that express receptors at physiological levels. To analyze the effect of PKC inhibitors on C5a-induced phosphorylation of C5aR1⁄7Ser-334, RBL-C5aR cells were pretreated for 30 min at 37 °C with the indicated concentrations of bisindolylmaleimide, rottlerin, LY333531, or Go6976.
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