MOR Phosphorylation in Opioid Tolerance and Dependence

描述(申请人提供)：激动剂诱导的阿片受体磷酸化被认为是一个重要的受体调节过程，促进急性受体脱敏，触发受体内化和再循环，并参与耐受的形成。大量来自表达克隆阿片受体的细胞系研究的实验证据表明，一般来说，MOR的磷酸化程度取决于配体的效力，但也可能受到类似效力的配体(例如，吗啡与美沙酮)和不同细胞中特定信号分子(CHO与HEK)的不同影响。这意味着MOR的磷酸化可能在不同的大脑区域具有不同的作用。然而，这还没有在神经系统和整个动物中得到证实。因此，这项研究旨在验证以下假设：MOR的磷酸化是激动剂依赖的，也是区域或细胞特异性的，受体的微环境和细胞信号成分影响磷酸化的程度和体内差异耐受和依赖的形成。以原代培养的DRG神经元为模型，PI建议检测DRG神经元和其他脑区的MOR磷酸化；评估其通过使用磷酸化缺陷的突变受体在脱敏和其他受体调节过程中的作用；以及确定细胞特异性MU磷酸化是否是DRG和其他脑区不同信号成分(如不同的蛋白激酶)参与的结果。PI还建议确定受体二聚化对MOR磷酸化的影响，探索受体二聚化对受体磷酸化的潜在区域差异和阿片耐受性发展的可能贡献。PI进一步建议通过使用敲入方法产生MOR磷酸化缺陷突变小鼠来改变动物的MOR磷酸化状态，从而在体内检验这一假说。因此，这些结果可能有助于更好地理解神经元中MOR磷酸化和其他受体调节过程之间的动态相互作用，并为阿片类药物耐受和依赖的不同发展机制提供潜在的解释，并为更有效的治疗提供新的基础。

DESCRIPTION (provided by applicant): Agonist-induced opioid receptor phosphorylation is believed to be an important receptor regulatory process that promotes acute receptor desensitization, triggers internalization and recycling of the receptors, and participates the development of tolerance. Numerous lines of experimental evidence from studies of cell lines that express cloned opioid receptor indicate that in general the extent of MOR phosphorylation is dependent on the ligand efficacy, but also could be influenced differentially by ligands with similar efficacy (morphine vs methadone, for example) and specific signaling molecules in the different cells (CHO vs HEK). This implies that MOR phosphorylation could have differential roles in different brain regions. However, this has not been demonstrated in neuronal systems and whole animals. Therefore, the proposed research is aimed at testing the following hypothesis: MOR phosphorylation is agonist-dependent as well as region or cell specific, and the receptor's microenvironment and cellular signaling components influence the extent of phosphorylation and the development of differential tolerance and dependence in vivo. Using primary culture of DRG neurons as a model, the PI proposes to examine the MOR phosphorylation in DRG neuron and other brain regions; to evaluate its contribution to desensitizationin and other receptor regulatory processes by using phosphorylation-deficient mutant receptors; and to determine if cellular specific mu phosphorylation is a result of participation of different signaling components (such as different protein kinases) among the DRG and other brain regions. The PI also proposes to determine the effects of receptor dimerization on MOR phosphorylation, exploring the possible contributions of receptor dimerization to the potential regional variation in receptor phosphorylation and the development of opioid tolerence. The PI further proposes to test the hypothesis in vivo by modifying the MOR phosphorylation status in animal through generating MOR phosphorylation-deficient mutant mice using a knock-in approach. Thus, the results could lead to a better understanding the dynamic interplay between MOR phosphorylation and other receptor regulatory processes in neurons, and to provide potential explanations on the mechanism of differential development of tolerance and dependence among opioid drugs and new foundations for more effective therapies.