Phosphorylation of C3a Receptor at Multiple Sites Mediates Desensitization, β-Arrestin-2 Recruitment and Inhibition of NF-κB Activity in Mast Cells

Background: Phosphorylation of G protein coupled receptors (GPCRs) by G protein coupled receptor kinases (GRKs) and the subsequent recruitment of beta-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and beta-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that beta-arrestin-2 provides an inhibitory signal for NF-kappa B activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, beta-arrestin recruitment and NF-kappa B activation has not been determined. Methodology/Principal Findings: We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on beta-arrestin-2 recruitment and RBL-2H3 cells for functional studies. We found that although Ala substitution of Ser475/479, Thr480/481 residues resulted in 58 +/- 3.8% decrease in agonist-induced C3aR phosphorylation there was no change in beta-arrestin-2 binding or receptor desensitization. By contrast, Ala substitution of Thr463, Ser465, Thr466 and Ser470 led to 40 +/- 1.3% decrease in agonist-induced receptor phosphorylation but this was associated with 74 +/- 2.4% decreases in beta-arrestin-2 binding, significantly reduced desensitization and enhanced NF-kappa B activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and beta-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater Ca2+ mobilization, degranulation and NF-kappa B activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of beta-arrestin (R169E) inhibited C3a-induced degranulation by 28 +/- 2.4% and blocked NF-kappa B activation by 80 +/- 2.4%. Conclusion/Significance: This study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, beta-arrestin-2 recruitment and inhibition of NF-kappa B activity. Furthermore, beta-arrestin-2 inhibits C3a-induced NF-kappa B activation via receptor desensitization-dependent and independent pathways.