G PROTEINS AND OPIOID RECEPTOR FUNCTIONS

描述：（申请人提供） 此K 05奖励申请的目的是让校长 调查员将注意力集中在他正在进行的研究项目上， 定期休假，从他的行政和教学 明尼苏达大学的承诺。K 05奖将允许PI花费 在他的合作者的实验室里， 接近他的研究目标。PI的职业目标是 阐明阿片耐受和依赖的分子机制。 对阿片类药物重复使用的耐受性和依赖性可能是 细胞对信号的补偿反应的结果是 由受体转导。因此，至关重要的是， PI实验室的总体目标，即，对机制的理解 其中神经元细胞可以整合膜传递的信号， 受体利用相同的第二信使系统谱。我们 以前的研究已经证明，克隆的阿片受体偶联， 以相似的效力激活Gi/Go蛋白。我们还建立了 μ阿片受体和δ阿片受体之间存在明显的差异， 调节相同的第二信使，如腺苷酸环化酶。的 可能存在的参与细胞蛋白质以外的 异源三聚体G蛋白在阿片受体信号传导中的作用。这是我们的 假设μ-和δ-阿片受体利用不同G蛋白 同样的效应器的调节，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， activation.阿片受体信号传导将涉及支架， 细胞蛋白质通过募集细胞蛋白质，如RGS， 在受体信号复合物中，信号的幅度和持续时间 可以被调制。信号的范围将取决于 信号复合物。因此，在目前的建议中，我们将使用 蜕皮激素可诱导的表达系统，以改变各种G蛋白， 亚基水平，以证明参与μ- 和δ-阿片受体信号。我们将使用随机饱和度 突变分析以及受体选择和扩增 技术（RSAT），以查明涉及μ-和δ-阿片类药物的领域 受体-G蛋白相互作用和激活。我们将展示现有的 阿片受体信号单位，通过蛋白质支架和 将识别出参与支架的细胞蛋白质。然后我们将 通过诱导型表达系统改变信号单元的含量， 研究这种改变对阿片受体信号传导的影响。这些 这些研究应该增强我们对阿片样物质的细胞调节的理解， 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.