Mann Anika, Illing Susann, Miess Elke, Schulz Stefan
Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
Br J Pharmacol. 2015 Jan;172(2):311-6. doi: 10.1111/bph.12627. Epub 2014 Jul 1.
The efficiency of μ-opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific μ-opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist-induced phosphorylation of μ-opioid receptors occurs at a conserved 10 residue sequence (370) TREHPSTANT(379) in the receptor's carboxyl-terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high-efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist-independent heterologous μ-opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite-specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high-efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of Gq -coupled receptors is mediated by PKCα. Rapid μ-opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ-opioid receptor phosphorylation. However, it remains to be seen to what extent different μ-opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo.
This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.
μ-阿片受体信号传导的效率受到严格调控，最终受细胞内丝氨酸和苏氨酸残基协同磷酸化的限制。在此，我们回顾并讨论磷酸化位点特异性μ-阿片受体抗体生成与应用方面的最新进展，这些抗体已被证明是监测受体磷酸化和去磷酸化时空动态的优秀工具。激动剂诱导的μ-阿片受体磷酸化发生在受体羧基末端细胞质尾部一个保守的10个残基序列（370）TREHPSTANT（379）处。多种阿片类药物在该序列中间的S375处诱导受体磷酸化，但只有高效阿片类药物有能力驱动侧翼残基（T370、T376和T379）的高阶磷酸化。S375是分级磷酸化级联反应中的起始残基。相比之下，非激动剂依赖性异源μ-阿片受体磷酸化主要发生在T370处。磷酸化位点特异性抗体与小干扰RNA敲低筛选相结合也有助于鉴定相关的激酶和磷酸酶。事实上，吗啡诱导的选择性S375磷酸化主要由G蛋白偶联受体激酶5（GRK5）催化，而高效阿片类药物诱导的多位点磷酸化特别需要GRK2/3。相比之下，佛波酯或Gq偶联受体异源激活刺激的T370磷酸化由蛋白激酶Cα（PKCα）介导。μ-阿片受体的快速去磷酸化发生在质膜处或其附近，由蛋白磷酸酶1γ（PP1γ）催化。这些发现表明，μ-阿片受体磷酸化的同源和异源调节存在不同的磷酸化基序。然而，不同的μ-阿片受体磷酸化模式在体内对耐受性和依赖性发展的贡献程度还有待观察。
本文是关于阿片类药物：功能选择性新途径主题部分的一部分。要查看本部分的其他文章，请访问http://dx.doi.org/10.1111/bph.2015.172.issue-2。
