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 β-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and β-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that β-arrestin-2 provides an inhibitory signal for NF-κB activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, β-arrestin recruitment and NF-κB activation has not been determined.


RESULTS
We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on β-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 β-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 β-arrestin-2 binding, significantly reduced desensitization and enhanced NF-κB activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and β-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater Ca(2+) mobilization, degranulation and NF-κB activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of β-arrestin (R169E) inhibited C3a-induced degranulation by 28±2.4% and blocked NF-κB activation by 80±2.4%.


CONCLUSIONS
This study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity. Furthermore, β-arrestin-2 inhibits C3a-induced NF-κB activation via receptor desensitization-dependent and independent pathways.
Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Kshitij
K
Gupta
Hariharan
H
Subramanian
Andreas
A
Klos
Hydar
H
Ali
D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism*; D015926:Complement C3a / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008407:Mast Cells / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011951:Receptors, Complement / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D016328:NF-kappa B; D011951:Receptors, Complement; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D015926:Complement C3a