Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists
Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists
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DOI：
10.1126/scisignal.aau8072
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发表时间：
2019-03-26
影响因子：
7.3
通讯作者：
Schulz, Stefan
中科院分区：
文献类型：
--
作者：
Mann, Anika;Mouledous, Lionel;Schulz, Stefan
痛觉肽/孤啡肽FQ阿片肽（NOP）受体激动剂是阿片受体家族的一员，作为新型镇痛药正在积极研究中，但它们的信号传导模式不像阿片受体家族的其他成员那样清晰。因此，我们研究了不同的NOP受体配体在NOP受体上是否表现出不同的信号传导或功能选择性。利用新开发的针对NOP受体的磷酸基特异性抗体，我们发现激动剂诱导的NOP受体磷酸化主要发生在四个羧基末端丝氨酸（Ser）和苏氨酸（Thr）残基上，即Ser（346）、Ser（351）、Thr（362）和Ser(363)，并且具有时间层次，Ser（346）是第一个磷酸化位点。G蛋白偶联受体激酶2和3 （GRK2/3）在激动剂诱导的磷酸化过程中协同作用，进而促进NOP受体脱敏和内化。结构上不同的NOP受体激动剂的比较揭示了G蛋白依赖信号和受体磷酸化之间的功能功效解离。此外，在NOP- egfp和NOP- eyfp小鼠中，NOP受体激动剂以剂量依赖和激动剂选择性的方式诱导多位点磷酸化和内化，可被特异性拮抗剂阻断。我们的研究为体内和体外研究配体激活的NOP受体信号提供了新的工具。不同化学类型的NOP受体激动剂对NOP受体的选择性磷酸化提示NOP受体激动剂的差异信号传导可能在NOP受体配体药理学中发挥作用。
Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely, Ser(346), Ser(351), Thr(362), and Ser(363), and proceeded with a temporal hierarchy, with Ser(346) as the first site of phosphorylation. G protein-coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which, in turn, facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein-dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.