Distinct and Shared Roles of β-Arrestin-1 and β-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

Background: The complement component C3a induces degranulation in human mast cells via the activation of cell surface G protein coupled receptors (GPCR; C3aR). For most GPCRs, agonist-induced receptor phosphorylation leads to the recruitment of beta-arrestin-1/beta-arrestin-2; resulting in receptor desensitization and internalization. Activation of GPCRs also leads to ERK1/2 phosphorylation via two temporally distinct pathways; an early response that reflects G protein activation and a delayed response that is G protein independent but requires beta-arrestins. The role of b-arrestins on C3aR activation/regulation in human mast cells, however, remains unknown. Methodology/Principal Findings: We utilized lentivirus short hairpin (sh) RNA to stably knockdown the expression of beta-arrestin-1 and beta-arrrestin-2 in human mast cell lines, HMC-1 and LAD2 that endogenously expresses C3aR. Silencing beta-arrestin-2 attenuated C3aR desensitization, blocked agonist-induced receptor internalization and rendered the cells responsive to C3a for enhanced NF-kappa B activity as well as chemokine generation. By contrast, silencing beta-arrestin-1 had no effect on these responses but resulted in a significant decrease in C3a-induced mast cell degranulation. In shRNA control cells, C3a caused a transient ERK1/2 phosphorylation, which peaked at 5 min but disappeared by 10 min. Knockdown of beta-arrestin-1, beta-arrestin-2 or both enhanced the early response to C3a and rendered the cells responsive for ERK1/2 phosphorylation at later time points (10-30 min). Treatment of cells with pertussis toxin almost completely blocked both early and delayed C3a-induced ERK1/2 phosphorylation in beta-arrestin1/2 knockdown cells. Conclusion/Significance: This study demonstrates distinct roles for beta-arrestins-1 and beta-arrestins-2 on C3aR desensitization, internalization, degranulation, NF-kappa B activation and chemokine generation in human mast cells. It also shows that both beta-arrestin-1 and beta-arrestin-2 play a novel and shared role in inhibiting G protein-dependent ERK1/2 phosphorylation. These findings reveal a new level of complexity for C3aR regulation by beta-arrestins in human mast cells.