MOR Phosphorylation in Opioid Tolerance and Dependence

描述（由申请人提供）：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，可以促进急性受体脱敏，触发受体的内化和再循环，并参与耐受性的发展。来自表达克隆阿片受体的细胞系研究的大量实验证据表明，一般来说，MOR磷酸化的程度取决于配体的功效，但也可能受到具有相似功效的配体（例如吗啡与美沙酮）和不同细胞中的特定信号分子（CHO与HEK）的差异影响。这意味着MOR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，本研究旨在验证以下假设：MOR磷酸化是激动剂依赖性的，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以及体内差异耐受和依赖性的发展。以DRG神经元原代培养为模型，PI提出检测DRG神经元和其他脑区MOR磷酸化；通过磷酸化缺陷突变受体评估其对脱敏蛋白和其他受体调节过程的贡献；并确定细胞特异性mu磷酸化是否是DRG和其他脑区之间不同信号成分（如不同蛋白激酶）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变化和阿片耐受性的发展的可能贡献。该团队进一步提出在体内验证这一假设，通过使用敲入方法产生MOR磷酸化缺陷突变小鼠，从而改变动物的MOR磷酸化状态。因此，该结果可以更好地理解神经元中MOR磷酸化与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性和依赖性的差异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.