G Proteins and Opiate Receptor Functions

描述（由申请人提供）：在过去的5年中，PI能够利用目前的K 05奖在他的实验室开发两个新的NIDA资助项目，并能够在加州大学洛杉矶分校的Chris Evans博士实验室度过一个学期的假期，探索使用斑马鱼作为研究阿片类药物耐受性和依赖性的分子机制的替代模型，这是PI的终身职业目标。这是可能的，因为从他的行政和教学承诺在明尼苏达大学由于K 05奖救济。因此，本K 05奖申请的目的仍然是一种机制，允许PI继续他的成功计划，从明尼苏达大学定期休假，并在他的合作者的实验室中花费时间，在研究耐受性和依赖性的分子机制方面追求新的或替代的方法和技术。从PI实验室正在进行的项目中可以清楚地看出，阿片受体通过受体体的形成发出信号。阿片受体与细胞蛋白质在微区中的支架作用对受体信号传导有很大影响。通过将不同的蛋白质募集到受体附近，β-arrestin、Src、RGS和AGS等信号的强度和持续时间均受到调制。PI已启动研究以识别可以调节阿片受体活性的细胞蛋白质。利用酵母双杂交筛选小鼠脑文库，发现FK 506结合蛋白FKBP 12能特异性地与莫尔的羧基尾部结构域相互作用，调节激动剂诱导的细胞内Ca 2+运动。然而，特定受体结构域的使用限制了需要多个结构域或三级受体结构用于结合的蛋白质的鉴定。因此，我们将继续我们正在进行的项目，以确定阿片受体体的组成部分，通过使用蛋白质组学方法和酵母双杂交筛选使用整个受体蛋白。用腺病毒过表达这些蛋白质，或通过siRNA敲低神经母细胞瘤N2 A细胞中的这些蛋白质水平，以确定它们对阿片受体调节的两种效应物的作用，即，腺苷酸环化酶和细胞内Ca 2+稳态。还将在富含表达内源性莫尔的神经元的原代海马培养物中进行这些蛋白质水平的改变。将开发诱导型siRNA载体并用于暂时敲低参与受体体形成的蛋白质。将在小鼠和其他模型中进行蛋白质水平的遗传改变，以测试这些蛋白质在慢性阿片类药物作用中的作用。

DESCRIPTION (provided by applicant): During the last 5 years, PI was able to utilize the current K05 award to develop two new NIDA funded projects in his laboratory, and was able to spend a semester leave in Dr. Chris Evans' laboratory at UCLA to explore the use zebrafish as an alternative model for studying the molecular mechanism of opiate tolerance and dependence, a life-long career goal of PI. This was made possible because of the relief from his administrative and teaching commitments at University of Minnesota due to the K05 award. Therefore, the objective of this K05 award application remains to be a mechanism allowing PI to continue his successful program of taking periodic leaves of absence from University of Minnesota and spend time in his collaborator's laboratory in the pursuit of new or alternative approaches and technologies in studying the molecular mechanism of tolerance and dependence. It is clear from the on-going projects in PI's laboratory that opioid receptor signals via the formation of receptorsomes. The scaffolding of cellular proteins with opioid receptor within the microdomains greatly affect the receptor signaling. By recruiting different proteins to the receptor vicinity, e.g., beta-arrestin, Src, RGS and AGS, the magnitude and duration of signals could be modulated. PI has initiated studies to identify the cellular proteins that could modulate opioid receptor activities. Using yeast two-hybrid screens of mouse brain library, protein candidates, such as the FK506 binding protein FKBP12 that specifically interacts with the carboxyl tail domain of MOR could modulate the agonist-induced intracellular Ca2+ movement. However, the use of a specific receptor domain limits the identification of proteins that require multiple domains or tertiary receptor structure for binding. Thus we will continue our on-going projects to determine the components of opioid receptorsomes by the use of proteomic approaches and yeast two-hybrid screens using whole receptor protein. Over-expression of these proteins with adenoviruses, or the knockdown of these proteins levels in neuroblastoma N2A cells by siRNA will be carried out to determine their effects on two the effectors regulated by opioid receptors, i.e., adenylyl cyclase and intracellular Ca2+ homeostasis. The alteration of these proteins levels in primary hippocampal cultures enriched in neurons expressing endogenous MOR will be carried out also. The inducible siRNA vector will be developed and used in the temporal knockdown of the proteins involved in the receptorsomes formation. Genetic alteration of the proteins levels will be carried out in mice and other models to test the effects of these proteins in chronic opiate drug actions.