G Proteins and Opiate Receptor Functions

描述(申请人提供)：在过去的5年里，Pi利用当前的K05奖在他的实验室开发了两个由NIDA资助的新项目，并能够在加州大学洛杉矶分校的Chris Evans博士的实验室度过一个学期的假期，探索使用斑马鱼作为研究鸦片耐受和依赖的分子机制的替代模型，这是Pi的终身职业目标。这是因为由于K05奖，他在明尼苏达大学的行政和教学承诺得到了缓解。因此，K05奖项申请的目标仍然是一种机制，允许Pi继续他的成功计划，从明尼苏达大学定期休假，并花时间在他的合作者的实验室里，追求新的或替代的方法和技术，研究耐受和依赖的分子机制。从Pi实验室正在进行的项目中可以清楚地看到，阿片受体是通过形成受体小体来传递信号的。微域内阿片受体与细胞蛋白的支架作用对受体信号转导有很大影响。通过在受体附近招募不同的蛋白质，如β-arrestin、Src、RGS和Ags，可以调节信号的大小和持续时间。PI已经开始研究，以确定可能调节阿片受体活性的细胞蛋白。利用酵母双杂交筛选小鼠脑文库，候选蛋白，如FK506结合蛋白FKBP12，特异性地与MOR的羧基尾部结构域相互作用，可以调节激动剂诱导的细胞内钙运动。然而，使用特定的受体结构域限制了对需要多个结构域或三级受体结构才能结合的蛋白质的识别。因此，我们将继续我们正在进行的项目，通过使用蛋白质组学方法和使用完整受体蛋白的酵母双杂交筛选来确定阿片受体小体的成分。用腺病毒过表达这些蛋白，或通过siRNA下调这些蛋白在神经母细胞瘤N2A细胞中的水平，以确定它们对阿片受体调控的两个效应器，即腺苷环化酶和细胞内钙稳态的影响。在原代培养的富含表达内源性MOR的神经元的海马区，这些蛋白质水平的变化也将被进行。可诱导的siRNA载体将被开发并用于在时间上击倒参与受体小体形成的蛋白质。这些蛋白质水平的遗传改变将在小鼠和其他模型中进行，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.