Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells.
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...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3471852?pdf=render |
| _version_ | 1818173728531415040 |
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| author | Kshitij Gupta Hariharan Subramanian Andreas Klos Hydar Ali |
| author_facet | Kshitij Gupta Hariharan Subramanian Andreas Klos Hydar Ali |
| author_sort | Kshitij Gupta |
| collection | DOAJ |
| description | 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.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%.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. |
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| format | Article |
| id | doaj.art-a4e04eb236384fadb3e1f7c53b8d65f7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T19:33:07Z |
| publishDate | 2012-01-01 |
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| spelling | doaj.art-a4e04eb236384fadb3e1f7c53b8d65f72022-12-22T00:53:13ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01710e4636910.1371/journal.pone.0046369Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells.Kshitij GuptaHariharan SubramanianAndreas KlosHydar AliPhosphorylation 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.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%.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.http://europepmc.org/articles/PMC3471852?pdf=render |
| spellingShingle | Kshitij Gupta Hariharan Subramanian Andreas Klos Hydar Ali Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells. PLoS ONE |
| title | Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells. |
| title_full | Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells. |
| title_fullStr | Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells. |
| title_full_unstemmed | Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells. |
| title_short | Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells. |
| title_sort | phosphorylation of c3a receptor at multiple sites mediates desensitization β arrestin 2 recruitment and inhibition of nf κb activity in mast cells |
| url | http://europepmc.org/articles/PMC3471852?pdf=render |
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