Molecular Basis of mu-Opioid Receptor Gene Regulation

描述(由申请人提供)：这是一份修订后的R01建议书(1 R01 DA016673-01)，是一名新的调查员。阿片受体(MOR)在镇痛以及耐受和依赖的形成中的关键作用已得到充分证实。然而，关于卫生部的监管仍有许多需要了解的地方。这个新的R01提案的目的是研究细胞特异性MOR基因表达的机制，特别是单链(Ss)DNA结合蛋白的作用及其与双链(DS)DNA结合蛋白在MOR基因调控中的功能相互作用。我们之前已经确定了一个远端和近端的MOR启动子，后者优先指导大脑中MOR的表达。在最近的启动子区域已经鉴定了几个顺式元件，包括DS和ASS顺式元件。Sp(ds结合因子)和一种未知的ss DNA结合蛋白也被证明参与其中，该蛋白特异性地与Mor ss顺式元件结合。这些数据为MOR基因如何在转录水平上进行调控提供了线索。最近，我们利用酵母单杂交筛选系统，成功地从小鼠脑cDNA文库中克隆了ASS DNA结合蛋白-聚C结合蛋白(PCBP)。我们的初步研究表明，该克隆的PCBP能高亲和力地与MOR顺式元件特异性结合，可能参与MOR基因的调控。PCBP从来没有作为一种转录因子被记录下来，它的目标基因也没有被确定。本研究将探讨PCBP作为转录调控因子在MOR基因表达调控中的新作用及其与其他转录因子的相互作用。首先将研究PCBP在调节MOR基因表达中的体内功能作用(具体目标1)。PCBP作为一种新的转录调控因子在MOR基因调控中的分子基础将在特定目标2中进行研究。PCBP和Sp转录因子在MOR基因调控中的相互作用以及Sp蛋白和PCBP的翻译后修饰对MOR基因调控的影响将在特定目标3中进行研究。最后，在特定目标4中，我们将研究PCBP是否直接与转录机制(包括RNA聚合酶II和辅助转录因子，如TFIID)相互作用来调节MOR基因的表达。从这些研究中获得的信息将为MOR基因调控的分子基础提供新的见解。对MOR基因表达调控机制的了解可能有助于开发改变受体表达的方法，并可能进一步帮助开发理解疼痛和药物滥用的新方法。

DESCRIPTION (provided by applicant): This is a revised R01 proposal (1 R01 DA016673-01) of a new investigator. The critical roles of the mu-opioid receptor (MOR) in analgesia, as well as in the development of tolerance and dependence are well-established. However, much remains to be learned about MOR regulation. The goal of this new R01 proposal is to investigate the mechanisms underlying cell-specific MOR gene expression, especially focusing on the roles of single-stranded (ss) DNA binding protein and its functional interaction with double-stranded (ds) DNA binding proteins in MOR gene regulation. We have previously identified a distal and a proximal MOR promoter, with the latter preferentially directing MOR expression in the brain. Several cis-elements have been identified, including the ds and ass cis-elements, in the proximal promoter region. The involvement of both Sp (ds binding factors) and an unidentified ss DNA binding protein, which specifically binds to the MOR ss ciselement, has also been shown. These data provided clues as to how the MOR gene is regulated at the transcriptional level. Recently, we have successfully cloned ass DNA binding protein, poly C binding protein (PCBP), from a mouse brain cDNA library using the yeast one-hybrid screening system. Our preliminary studies strongly suggested that this cloned PCBP can specifically bind to the MOR ss cis-element with high affinity, and may participate in the MOR gene regulation. PCBP has never been documented as a transcription factor, nor has its target gene been identified. The novel role of PCBP as a transcription regulator as well as its interaction with other transcription factors in the regulation of MOR gene expression will be examined in this proposal. The in vivo functional roles of PCBP in the regulation of MOR gene expression will first be examined (Specific Aim 1). The molecular basis of how PCBP can serve as a novel transcriptional regulator in MOR gene regulation will be examined in Specific Aim 2. The interplay (interaction) between PCBP and Sp transcription factors in regulation of MOR gene, as well as the effects of post-translational modifications of Sp proteins and PCBP on MOR gene regulation, will be examined in Specific Aim 3. Finally, in Specific Aim 4, we will examine whether PCBP directly interacts with the transcriptional machinery (including RNA polymerase II and auxiliary transcription factors, such as TFIID) in regulating MOR gene expression. The information gained from these studies will provide new insights into the molecular basis of MOR gene regulation. The understanding of mechanisms regulating MOR gene expression may help in the development of approaches to alter the receptor expression and may further help in the development of novel approaches understanding pain as well as drug abuse.