MOR Phosphorylation in Opioid Tolerance and Dependence

描述（由申请方提供）：激动剂诱导的阿片受体磷酸化被认为是一种重要的受体调节过程，可促进急性受体脱敏，触发受体的内化和再循环，并参与耐受性的形成。来自表达克隆的阿片样物质受体的细胞系的研究的大量实验证据表明，一般来说，莫尔磷酸化的程度取决于配体功效，但也可能受到具有相似功效的配体（例如吗啡对美沙酮）和不同细胞中的特异性信号传导分子（CHO对HEK）的不同影响。这意味着莫尔磷酸化在不同的脑区可能具有不同的作用。然而，这尚未在神经元系统和整个动物中得到证实。因此，所提出的研究旨在检验以下假设：莫尔磷酸化是激动剂依赖性的以及区域或细胞特异性的，并且受体的微环境和细胞信号传导组分影响磷酸化的程度以及体内差异耐受性和依赖性的发展。PI计划以原代培养的DRG神经元为模型，检测DRG神经元和其他脑区的莫尔磷酸化，利用磷酸化缺陷突变受体评价其对脱敏和其他受体调节过程的贡献;并确定细胞特异性μ磷酸化是否是不同信号传导组分参与的结果（如不同的蛋白激酶）之间的DRG和其他大脑区域。PI还建议确定受体二聚化对莫尔磷酸化的影响，探索受体二聚化对受体磷酸化潜在区域变化和阿片耐受性发展的可能贡献。PI进一步提出通过使用敲入方法产生莫尔磷酸化缺陷突变小鼠，通过改变动物中的莫尔磷酸化状态，在体内检验该假设。因此，这些结果可能会导致更好地了解莫尔磷酸化和其他受体调节过程之间的动态相互作用，并提供阿片类药物之间的耐受性和依赖性的差异发展的机制和更有效的治疗新的基础提供潜在的解释。

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.