Dynamics of protein kinase C mediated phosphorylation of the C5a receptor on Serine‐334

Abstract

Phosphorylation of Ser-334, one out of three key serine phosphorylation sites within the C5aR carboxyl terminus, was shown to be required for the C5a-stimulated internalization of this chemotactic leukocyte receptor (Braun et al., J Biol Chem 278:4277, 2003). Because of the functional significance of this phosphorylation site for C5aR endocytosis we generated phosphosite-specific monoclonal antibodies which specifically react with phosphoserine-334 in ligand-activated C5aR. Treatment of db-cAMP stimulated U937 cells, which endogenously express C5aR, with activators (PMA) or inhibitors (bisindolylmaleimide) of protein kinase C (PKC) revealed that both homologous, C5a-induced and heterologous, C3a-induced phosphorylation of Ser-334 was mediated by PKC. A C5aR synthetic peptide was found to be a substrate for recombinant conventional (α,β I, β II, γ) and novel (δ) PKC isozymes in an in vitro phosphorylation assay. However, in intact myeloid cells only a PKCβ I/β II-selective inhibitor (LY333531), but not a PKCδ inhibitor efficiently blocked Ser-334 phosphorylation. Consistent with our previous observation in the related CCR5 receptor system, PKC phosphorylated C5aR at low C5a concentrations in a very rapid (t½ ~ 20 sec), albeit transient manner. Surprisingly, at high concentrations (>10nM) of C5a the PKC-mediated phosphorylation of serine-334 was essentially blocked. This could be attributed to the even faster (t½ < 5 sec) GRK-mediated phosphorylation of C5aR and consecutive firm binding of ß-arrestin. Our findings indicate that β-arrestin modulates PKC-mediated receptor phosphorylation and thus define a new regulatory role for this important adaptor protein.