Regulation of opioid tolerance by agonist-selective phosphorylation of endogenous µ-opioid-receptors in vivo

吗啡仍然是治疗剧烈疼痛最有效的药物之一。然而，吗啡和其他阿片类止痛药在治疗慢性疼痛方面的临床益处受到耐受性和依赖性的快速发展的限制。吗啡的所有药理作用都是通过G蛋白偶联的Mu阿片受体(MOR)介导的。磷酸化是调节G蛋白偶联受体(GPCRs)的一个重要机制，主要是通过改变它们与不同细胞内伙伴相互作用的能力来实现的。激动剂诱导的GPCRs的磷酸化是由一系列G蛋白偶联受体激酶(GRKs)介导的，GRKs特异性地识别这些受体的激活状态。我们以前已经证明，激动剂诱导的MOR的磷酸化发生在羧基末端细胞质尾部的一个保守的10个残基序列370TREHPSTANT379上。吗啡诱导序列中间的丝氨酸375(S375)选择性磷酸化，主要由GRK5催化。相比之下，高效的阿片类药物，如DAMGO或芬太尼，不仅诱导S375的磷酸化，还推动侧翼残基苏氨酸370(T370)、苏氨酸376(T376)和苏氨酸379(T379)的高阶磷酸化，这是一个特定需要GRK2/3亚型的分级磷酸化级联反应。我们还表明，GRK2/3介导的磷酸化是导致MOR快速脱敏的原因，而GRK5是阿片类药物依赖形成过程中的一个重要因素。本研究的目的是：1)利用新的HAMOR敲门小鼠体内分析激动剂选择性的MOR磷酸化；2)阐明C端磷酸化在利用磷酸化缺陷的MOR敲击小鼠发展阿片耐受中的作用；3)利用新的GRK5-KD敲门小鼠研究GRK5在阿片依赖形成中的作用；以及4)确定异源PKC介导的MOR磷酸化在吗啡耐受形成中的作用。

Morphine is still one of the most effective drugs to treat severe pain. However, the clinical benefit of morphine and other opioid analgesics in the treatment of chronic pain is limited by the rapid development of tolerance and dependence. Morphine mediates all of its pharmacological effects via the G-protein-coupled mu-opioid receptor (MOR). Phosphorylation is an important mechanism for the regulation G protein-coupled receptors (GPCRs) mainly by altering their ability to interact with different intracellular partners. The agonist-induced phosphorylation of GPCRs is mediated by a family of G protein-coupled receptor kinases (GRKs) that specifically recognize the activated state of these receptors. We have previously shown that agonist-induced phosphorylation of MOR occurs at a conserved 10-residue sequence, 370TREHPSTANT379, in the carboxyl-terminal cytoplasmic tail. Morphine induces a selective phosphorylation of serine375 (S375) in the middle of this sequence that is predominantly catalyzed by GRK5. By contrast, high-efficacy opioids such as DAMGO or fentanyl not only induce phosphorylation of S375 but also drive higher-order phosphorylation on the flanking residues threonine370 (T370), threonine376 (T376), and threonine379 (T379) in a hierarchical phosphorylation cascade that specifically requires GRK2/3 isoforms. We have also shown that GRK2/3-mediated phosphorylation is responsible for rapid desensitization of MOR, while GRK5 is an essential element in the development of opioid dependence. The objectives of this proposal are 1) to analyze the agonist-selective phosphorylation of MOR in vivo using a novel HAMOR knockin mouse, 2) to elucidate the function of C-terminal phosphorylation in the development of opioid tolerance using phosphorylation-deficient MOR knockin mice, 3) to characterize the role of GRK5 in the development of opioid dependence using a novel GRK5-KD (kinase-dead) knockin mouse, and 4) to determine the contribution of heterologous PKC-mediated MOR phosphorylation to the development of morphine tolerance.