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JOURNAL ARTICLE
RESEARCH SUPPORT, U.S. GOV'T, P.H.S.
Binding of Sp1/Sp3 to the proximal promoter of the hMOR gene is enhanced by DAMGO.
Gene 2001 August 23
The major binding site for morphine is the mu opioid receptor (MOR), which mediates morphine's analgesic and euphoric effects. The MOR gene is highly regulated at the level of transcription. The present study examined DNA-protein interactions in the human MOR (hMOR) -500 to -292 promoter region, and tested whether chronic opioid drug treatment could modulate these DNA-protein interactions. 5'-deletion and transient transfection assays in SK-N-SH cells revealed four regions that activated hMOR gene transcription. A 60 bp sequence (-351 to -292) upstream of the proximal transcription initiation site (-252) contained cis-elements required for basal promoter activity. Sp1 and Sp3 bound to this 60 bp region, which was confirmed by electromobility shift assays using a Sp1 consensus oligo as competitor and specific antibodies against Sp1 and Sp3. Methylation interference analysis localized the Sp1 binding site to the sequence CCCTCCTCCC (-310 to -301) and also suggested that additional transcription factors, other than Sp1-related proteins, contacted the -321 to -301 sequence. Moreover, the binding of Sp1/Sp3 to the hMOR promoter was significantly enhanced by chronic exposure to [D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin (DAMGO), a selective MOR agonist, and this effect was attenuated specifically by pretreatment with a MOR antagonist, naloxone. Taken together, the present studies demonstrated, for the first time, that the binding of Sp1/Sp3 to the hMOR proximal promoter could be modulated by chronic DAMGO treatment. Such enhanced binding of Sp1/Sp3 to the promoter may lead to a functional change in hMOR gene transcription.
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