Phosphorylation patterns on the c-terminal tail of the angiotensin II type 1 receptor encode differential signaling pathways

Structural studies have recently shown that mechanism of selective AT1R activation by different classes of ligands is initiated by the ability of ligands to stabilize unique active conformations of the receptor. Active conformations enhance transducer coupling leading to effector mediated signaling, that has been proposed to be linked to differential phosphorylation of the c-terminal tail of the receptor. To determine how different classes of AT1R ligands effect receptor phosphorylation and activation of downstream signaling we used TMT-MS to identify the amino acid residues of the AT1R phosphorylated following treatment with the full balanced agonist Angiotensin II and a β-arrestin biased ligand, TRV023. Here we show that Ang and TRV 023 induce two distinct patterns of phosphorylation clusters a.a. residues along the entire c-tail with AngII inducing a greater magnitude than the β-arrestin biased ligand TRV 023, particularly in the proximal part of the tail. Selective mutagenesis of Ser and Thr residues, we found that of the 12 Ser/Thr residues within the c-tail, only the 4 proximal and 4 middle residues are all required for full β -arrestin functionality in response to either a balanced or β-arrestin-based ligand. Surprisingly, phosphorylation of 4 residues in the proximal c-tail is necessary for maximal G-protein activation to a full agonist. Taken together our data demonstrate that a AT1R ligands that stabilize different active confirmations of the receptor (full agonist vs. β-arrestin biased) induce distinct phosphorylation patterns on the c-tail of the receptor to evoke distinct receptor-transducer engagement and downstream receptor trafficking and signaling.

近期结构研究显示，不同类别配体对血管紧张素II 1型受体（AT1R）的选择性激活机制，始于配体稳定受体独特活性构象的能力。活性构象可增强转导蛋白偶联，进而介导效应分子的信号转导，该过程被认为与受体羧基末端尾部（C-terminal tail）的差异化磷酸化密切相关。为明确不同类别AT1R配体如何影响受体磷酸化及下游信号转导的激活，我们采用串联质谱标签-质谱（TMT-MS）技术，鉴定了经完全平衡激动剂血管紧张素II（Angiotensin II）与β抑制蛋白（β-arrestin）偏向性配体TRV023处理后，发生磷酸化的AT1R氨基酸残基。本研究结果表明，血管紧张素II（后文简称Ang II）与TRV023可在受体完整C端尾上诱导两种截然不同的磷酸化簇分布模式；其中Ang II诱导的磷酸化强度显著高于β抑制蛋白偏向性配体TRV023，在尾部近端区域尤为突出。通过对丝氨酸（Ser）与苏氨酸（Thr）残基的定向诱变实验，我们发现：在C端尾的12个Ser/Thr残基中，仅近端的4个与中部的4个残基，是响应平衡激动剂或β抑制蛋白偏向性配体时，实现完整β抑制蛋白功能所必需的。令人意外的是，C端尾近端4个残基的磷酸化，对于完全激动剂诱导的最大程度G蛋白（G-protein）激活是必不可少的。综合本研究所有数据，我们证实：稳定受体不同活性构象的AT1R配体（完全激动剂与β抑制蛋白偏向性配体），可在受体C端尾上诱导截然不同的磷酸化模式，进而引发差异化的受体-转导蛋白结合以及下游受体转运与信号转导过程。