Molecular Basis of mu-Opioid Receptor Gene Regulation

描述（由申请人提供）：这是一份新研究者的修订R 01提案（1 R 01 DA 016673 -01）。μ-阿片受体（莫尔）在镇痛以及耐受性和依赖性的发展中的关键作用是公认的。然而，关于莫尔的监管，仍有许多东西有待了解。这个新的R 01提案的目标是研究细胞特异性莫尔基因表达的潜在机制，特别关注单链（ss）DNA结合蛋白及其与双链（ds）DNA结合蛋白在莫尔基因调控中的功能相互作用。我们以前已经确定了一个远端和近端莫尔启动子，后者优先指导莫尔在大脑中的表达。在近端启动子区域已经鉴定出几个顺式元件，包括ds和ass顺式元件。还显示了Sp（ds结合因子）和特异性结合莫尔ss顺式元件的未鉴定的ss DNA结合蛋白的参与。这些数据为莫尔基因在转录水平上的调控提供了线索。最近，我们利用酵母单杂交筛选系统，从小鼠脑cDNA文库中成功地克隆了一个与DNA结合的蛋白--聚C结合蛋白（Poly C binding protein，PCBP）。初步研究表明，该PCBP能与莫尔还原酶ss顺式元件特异性结合，并可能参与了莫尔还原酶基因的调控。PCBP从未被记录为转录因子，也没有确定其靶基因。PCBP作为转录调节因子的新作用以及它与其他转录因子在调节莫尔基因表达中的相互作用将在本提案中进行检查。将首先检查PCBP在调节莫尔基因表达中的体内功能作用（具体目标1）。PCBP在莫尔基因调控中如何作为一种新型转录调节因子的分子基础将在具体目标2中进行研究。将在具体目标3中检查PCBP和Sp转录因子在莫尔基因调控中的相互作用（相互作用），以及Sp蛋白和PCBP的翻译后修饰对莫尔基因调控的影响。最后，在具体目标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，调节莫尔基因表达。从这些研究中获得的信息将为莫尔基因调控的分子基础提供新的见解。了解调节莫尔基因表达的机制可能有助于开发改变受体表达的方法，并可能进一步有助于开发了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.