Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells.
Phosphorylation of C3a receptor at multiple sites mediates desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity in mast cells.
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DOI：
10.1371/journal.pone.0046369
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Ali H
中科院分区：
文献类型：
--
作者：
Gupta K;Subramanian H;Klos A;Ali H
G蛋白偶联受体激酶（GRK）对G蛋白偶联受体（GPCR）的磷酸化以及随后的β-抑制蛋白的募集对于它们的脱敏是重要的。最近，我们使用shRNA介导的基因沉默策略，发现GRK 2、GRK 3和β-arrestin-2促进人肥大细胞C3 a受体（C3 aR）的脱敏。我们还证实了β-arrestin-2可抑制NF-κB的活化。C3 aR在其羧基端具有10个潜在的磷酸化位点，但它们在脱敏、β-arrestin募集和NF-κB活化中的作用尚未确定。我们在转染的HEK 293细胞中利用定点诱变方法来确定受体磷酸化对β-抑制蛋白-2募集和RBL-2 H3细胞的作用，以进行功能研究。我们发现，虽然Ala取代Ser 475/479，Thr 480/481残基导致激动剂诱导的C3 aR磷酸化降低58±3.8%，但β-arrestin-2结合或受体脱敏没有变化。相比之下，Thr 463、Ser 465、Thr 466和Ser 470的Ala取代导致激动剂诱导的受体磷酸化降低40±1.3%，但这与β-arrestin-2结合降低74±2.4%、显著降低脱敏和增强NF-κB活化相关。这些Ser/Thr残基的组合突变沿着Ser 459（突变MT 7），导致受体磷酸化和β-arrestin-2结合的完全丧失。与野生型受体相比，表达MT 7的RBL-2 H3细胞对C3 a的反应是更大的Ca 2+动员、脱粒和NF-κB活化。有趣的是，MT 7与β-抑制蛋白的组成型活性突变体（R169 E）的共表达抑制了28±2.4%的C3 a诱导的脱粒，并阻断了80± 2.4%的NF-κB活化。本研究表明，尽管C3 a在多个位点引起其受体的磷酸化，但Ser 459、Thr 463、Ser 465、Thr 466和Ser 470参与C3 aR脱敏、β-arrestin-2募集和NF-κB活性抑制。此外，β-arrestin-2通过受体脱敏依赖性和非依赖性途径抑制C3 a诱导的NF-κB活化。
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 Ca2+ 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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影响因子：
4.4
作者：
Ali, H;Ahamed, J;Patel, DD
通讯作者：
Patel, DD
Beta-arrestin1与G蛋白亚基β1GAMMA2相互作用，并促进beta1gamma2依赖性AKT信号，以用于NF-kappab激活。
DOI：
10.1042/bj20081561
发表时间：
2009-01-01
期刊：
The Biochemical journal
影响因子：
--
作者：
Yang M;He RL;Benovic JL;Ye RD
通讯作者：
Ye RD
影响因子：
5.4
作者：
Settmacher, B;Rheinheimer, C;Klos, A
通讯作者：
Klos, A
影响因子：
14.2
作者：
Schaefer, Beatrix;Piliponsky, Adrian M.;Oka, Tatsuya;Song, Chang Ho;Gerard, Norma P.;Gerard, Craig;Tsai, Mindy;Kalesnikoff, Janet;Galli, Stephen J.
通讯作者：
Galli, Stephen J.
影响因子：
4.8
作者：
Potter, RM;Key, TA;Prossnitz, ER
通讯作者：
Prossnitz, ER