Role of an AP-2-like element in transcriptional regulation of mouse mu-opioid receptor gene.
Previously, several important cis-elements and trans-factors have been shown to play a functional role in the proximal promoter of mouse mu-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.
Ko
Jane L
JL
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA. kojane@shu.edu
Liu
Hsien Ching
HC
Loh
Horace H
HH
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002490:Central Nervous System / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics*; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D018847:Opioid Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D017450:Receptors, Opioid, mu / Q000096:biosynthesis* / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C436492:KLF12 protein, human; D051741:Kruppel-Like Transcription Factors; D018847:Opioid Peptides; D017450:Receptors, Opioid, mu; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D017868:Cyclic AMP-Dependent Protein Kinases