G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of β-Arrestin
G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of β-Arrestin
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DOI：
10.1124/mol.116.106468
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发表时间：
2017-06-01
影响因子：
3.6
通讯作者：
Benovic, Jeffrey L.
中科院分区：
文献类型：
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作者：
Luo, Jiansong;Busillo, John M.;Benovic, Jeffrey L.
G蛋白偶联受体（GPCRs）的磷酸化是细胞信号传导和受体功能调节的关键事件。以前，使用串联质谱，我们确定了两个磷酸化位点在远端的C-末端尾部的趋化因子受体CXCR 4，但无法确定哪些特定的残基被磷酸化。在这里，我们证明了CXCR 4的丝氨酸（Ser）346和/或347（Ser-346/7）在用激动剂CXCL 12以及CXCR 4 pepducin ATI-2341刺激后被磷酸化。ATI-2341是一种G α 1 β γ异源三聚体偏向的CXCR 4激动剂，与CXCL 12相比，其诱导了更强的Ser-346/7磷酸化。G蛋白偶联受体激酶（GRK）2、GRK 3或GRK 6的敲低降低了CXCL 12诱导的Ser-346/7的磷酸化，其中GRK 3敲低具有最强的作用，而常规蛋白激酶C（PKC）同种型（特别是PKC α）的抑制降低了由CXCL 12或ATI-2341诱导的Ser-346/7的磷酸化。GRK 3或PKC介导的Ser-346/7磷酸化的丧失损害了b-抑制蛋白向CXCR 4的募集。我们还发现，PKC zeta的假底物肽抑制剂有效抑制CXCR 4磷酸化和信号传导，最有可能是作为非特异性CXCR 4拮抗剂发挥作用。总之，这些研究证明了Ser-346/7在抑制蛋白募集和启动受体脱敏中的作用，并提供了对在各种形式的WHIM综合征患者中观察到的CXCR 4失调的见解。
Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a G alpha i beta gamma heterotrimer-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKC alpha, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3-or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of b-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKC zeta effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.