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.
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