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Identification and Characterization of Two Nuclear Factor-B Sites in the Regulatory Region of the Dopamine D2 Receptor

Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies (S.B., C.F., C.B., N.G., P.F.S., C.M.), and Center of Excellence on Diagnostic and Therapeutic Innovation (P.F.S., C.M.), University of Brescia, 25124 Brescia, Italy

Address all correspondence and requests for reprints to: Cristina Missale, Ph.D., Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, University of Brescia, Viale Europa 11, 25124 Brescia, Italy. E-mail: cmissale{at}med.unibs.it.

Regulation of D2 receptor (D2R) expression is crucial in the function of dopaminergic systems. Because alterations of D2R expression may contribute to the development of different disorders, it is important to elucidate the mechanisms regulating D2R gene transcription. We report the characterization of two putative nuclear factor-B (NF-B) motifs, referred to as D2-B sites, in the human D2R promoter, and demonstrate that they bind NF-B subunits and stimulate D2R promoter activity. D2-B sites show different degrees of conservation and specificity, when compared with canonical kB sites. The D2-B1 site (from –407 to –398) is highly conserved and binds p50/p65 and p50/c-Rel complexes, whereas D2-B2 (from –513 to –504) is more degenerated and only binds p50/p65 heterodimers. Activation of D2-B sites in COS-7 cells expressing a luciferase reporter vector containing the D2R promoter resulted in increased transcriptional activity. Site-directed mutagenesis of each D2-B site differentially modified D2R promoter activity. In particular, mutation of the D2-B1 motif did not affect D2R promoter response to p50/c-Rel complexes, whereas inactivation of the D2-B2 site decreased it. Mutations of either D2-B1 or D2-B2 sites attenuated the D2R promoter transcriptional efficiency induced by p50/p65 complexes. Thus, D2R transcription mediated by p50/c-Rel is supported mainly by the D2-B2 site, whereas both sites are necessary to support the full transcriptional activity mediated by p50/p65 complexes. A correlation was found between NF-B activity and D2R expression in the pituitary and pituitary-derived cells but not in the striatum, suggesting that NF-B regulation of D2R expression could be a pituitary-specific mechanism.