G PROTEINS AND OPIOID RECEPTOR FUNCTIONS

描述：(申请人提供) 本K05奖项申请的目的是为了让校长 调查员将注意力集中在他正在进行的研究项目上，并 定期请假离开他的行政和教学工作 在明尼苏达大学的承诺。K05奖将允许少年派 花时间在合作者的实验室里，寻求新的或替代的 实现他的研究目标的方法。少年派的职业目标一直是 阐明阿片类药物耐受和依赖的分子机制。 对重复使用阿片类药物的耐受性和依赖性可能是 细胞对信号的代偿性反应的后果 由受体转导。因此，最重要的是解决 PI实验室的总体目标，即对机制的理解 其中神经细胞可以整合通过膜传递的信号 利用相同光谱的第二信使系统的受体。我们的 先前的研究表明，克隆的阿片受体偶联并 激活具有类似效力的Gi/Go蛋白。我们也已经建立了 Mu和Delta阿片受体之间有明显的区别 调节相同的第二信使，如腺苷环化酶。这个 存在细胞蛋白质参与的可能性，而不是 异三聚体G蛋白在阿片受体信号转导中的作用。因此，这是我们的 假设Mu-和Delta-阿片受体利用不同的G蛋白 对同一效应器的调节，这是由于微妙的 参与G蛋白相互作用的受体结构域内的差异和 激活。阿片受体信号转导将涉及 细胞蛋白质。通过将RGS等细胞蛋白招募到附近 受体信号复合体中，信号的幅度和持续时间 可以被调制。信号的大小将取决于 信号复合体。因此，在当前的提案中，我们将使用 蜕皮激素-哺乳动物诱导表达系统改变各种G蛋白a 亚基水平，以显示参与MU-1的特异性G蛋白。 和阿片受体信号转导。我们将使用随机饱和度 突变分析与受体选择和扩增 技术(RSAT)，以确定Mu-和Delta-阿片类药物涉及的领域 受体-G蛋白的相互作用和激活。我们将演示现有的 阿片受体信号单位中，通过蛋白质支架和 将鉴定参与脚手架的细胞蛋白质。到时候我们会的 通过可诱导表达系统改变信号单元的内容 研究这种改变对阿片受体信号的影响。这些 研究应该加强我们对阿片类药物细胞调节的理解 受体信号。

DESCRIPTION: (Provided by Applicant) The purpose of this K05 award application is to allow the principal investigator to focus his attention on his on-going research projects and to take periodic leaves of absence from his administrative and teaching commitments at University of Minnesota. The K05 award will allow PI to spend time in his collaborators' laboratories and pursue new or alternative approaches to his research goals. The career goal of PI has been the elucidation of the molecular mechanism of opioid tolerance and dependence. Tolerance and dependence to the repeated use of opioid drugs can be the consequences of the cellular compensatory responses to the signals being transduced by the receptors. Thus, it is of utmost importance to address an overall objective of PI's laboratory, i.e., the understanding of the mechanism in which neuronal cells could integrate the signals transduced by membrane receptors that utilize the same spectrum of second messenger systems. Our previous studies have demonstrated that the cloned opioid receptors coupled and activated the Gi/Go proteins with similar potencies. We have established also that there are distinct differences between mu and delta opioid receptor regulation of the same second messenger such as adenylyl cyclase. The probability exists for the involvement of cellular proteins other than the heterotrimeric G proteins in the opioid receptor signaling. Thus, it is our hypothesis that mu- and delta-opioid receptors utilize different G proteins for the regulation of the same effector, and that this is due to the subtle differences within the receptor domains involved in G protein interaction and activation. The opioid receptor signaling will involve the scaffolding of cellular proteins. By recruiting cellular proteins such as RGS to the proximity of the receptor signaling complexes, the amplitude and duration of the signals can be modulated. The extent of the signal will depend on the composition of the signaling complexes. Hence, in the current proposal, we will use the ecdysone mammalian-inducible expression system to alter the various G protein a subunit level so as to demonstrate the specific G protein involved in the mu- and delta-opioid receptor signaling. We will use the random saturation mutational analysis together with the Receptor Selection and Amplification Technology (RSAT) to pinpoint the domains involved in mu- and delta-opioid receptor-G protein interaction and activation. We will demonstrate the existing of opioid receptor signaling units, the signaling via protein scaffolding and will identify the cellular proteins involved in the scaffolding. We will then alter the contents of signaling units by the inducible expression system and examine the effects of such alteration on opioid receptor signaling. These studies should enhance our understanding of cellular regulation of the opioid receptor signaling.