Role of an AP-2-like element in transcriptional regulation of mouse μ-opioid receptor gene

Jane L. Ko, Hsien Ching Liu, Horace H Loh

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25 Scopus citations

Abstract

Previously, several important cis-elements and trans-factors have been shown to play a functional role in the proximal promoter of mouse μ-opioid receptor (MOR) gene. In this study, we defined another functional element located the in -450 to -400 bp (translational start site designated as +1) region of the proximal promoter, which is also essential for the full promoter activity. It is designated as the morAP-2-like element for its sequence homologous to the consensus AP-2 element. Surprisingly, electrophoretic mobility shift analysis (EMSA) revealed that Sp1 and Sp3, but not AP-2 proteins, were specifically bound to the morAP-2-like element. Mutation of the morAP-2-like element, resulting in a loss of Sp binding, led to an approximately 35% decrease in activity, further confirming the positive role of the morAP-2-like element in MOR gene expression. Dephosphorylation of Sp proteins with alkaline phosphatase also decreased Sp binding to the morAP-2-like element in EMSA, suggesting phosphorylation of Sp is essential for its binding to this element. However, direct or indirect activation of PKA, a classical G-protein coupled signaling pathway, resulted in no significant change of Sp binding to the morAP-2-like element, nor of the promoter activity the SH-SY5Y cells, MOR expressing cells, suggesting that phosphorylation of Sp does not involve PKA. These results suggest that the binding of different phosphorylated forms of Sp proteins to the morAP-2-like element may contribute to the fine tuning of MOR expression in different cells.

Original languageEnglish (US)
Pages (from-to)153-162
Number of pages10
JournalMolecular Brain Research
Volume112
Issue number1-2
DOIs
StatePublished - Apr 10 2003

Bibliographical note

Funding Information:
We thank Dr Andrew P. Smith for editing the manuscript, as well as Mrs Sharon R. Minnerath and Mr Guilin Wang for their excellent technical supports. This research was supported by NIH research grants DA-00564, DA-01583, P50-DA-11806, KO5-DA-70554, and A and F Stark Fund of the Minnesota Medical Foundation

Keywords

  • AP-2-like element
  • Mouse MOR promoter
  • Phosphorylation/dephosphorylation
  • Sp1/3

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