Abstract
BackgroundThe complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.Methodology/Principal FindingsWe utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca2+ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.Conclusion/SignificanceThis study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.
Highlights
The complement component C3a plays an important role in innate immunity and promotes allergic diseases such as bronchial asthma [1,2,3]
To determine the roles of G protein coupled receptor kinases (GRKs) on the regulation of C3aR signaling in human mast cells, we used the Mission shRNA lentiviral system to stably knockdown the expression of GRK2, GRK3, GRK5 or GRK6 in a human mast cell line, HMC-1 cells
Using real time quantitative PCR we found that the mRNA levels of GRK2 and GRK5 were reduced by .90% in HMC-1 cells (Fig. 1A and 1C)
Summary
The complement component C3a plays an important role in innate immunity and promotes allergic diseases such as bronchial asthma [1,2,3]. Removal of potential phosphorylation sites within the carboxyl terminus of C3aR leads to more robust degranulation when compared to wildtype receptors [7] These findings are consistent with the idea that, as in many other cell types, receptor phosphorylation desensitizes C3aR expressed in mast cells [8]. Studies with siRNA-mediated knockdown of GRKs in HEK293 cells have shown that agonist-induced phosphorylation of angiotensin II type 1A receptor (Gq-coupled) and V2 vasopressin receptors (Gscoupled) are predominantly mediated by GRK2 and GRK3 [10,11]. Knockdown of these GRKs attenuated both agonist-induced b-arrestin recruitment and receptor desensitization [10,11]. The roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown
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