SP FAMILY TRANSCRIPTION FACTORS REGULATE EXPRESSION OF RAT D-2 DOPAMINE-RECEPTOR GENE

The rat D-2 dopamine receptor gene is transcribed from a TATA-less pro moter that has an initiator-like sequence and several putative Sp1 bin ding sites. We previously reported that a negative modulator is locate d between nucleotides -116 and -76 (D(2)Neg-B) in this gene and that S p1 as well as another unknown factor bind to this region (Minowa et al ., J. Biol. Chem. 269, 11656, 1994). In the present investigation empl oying the in situ filter detection method, we identified this factor a s Sp3, Anti-Sp3 antiserum used in gel-shift assays also revealed that Sp3 binds to the D(2)Neg-B sequence. Cotransfection of Drosophila Schn eider's SL2 cells with Sp3 or Sp1 expression plasmids in the presence of CAT reporter plasmids containing D-2 promoter regions demonstrated that Sp1 increased CAT activity in a dose-dependent manner, whereas Sp 3, either alone or in the presence of Sp1, failed to activate or repre ss transcription. On the other hand, using the TATA-containing reporte r plasmid BCAT-2, Sp3 coexpression significantly repressed Sp1-induced trans-activation, although Sp3 alone was ineffective. Thus, the trans criptional activity of Sp3 is dependent on the promoter context, and t he negative regulation of D-2 gene expression appears quite complex an d mag not depend simply an known DNA-protein interactions involving on ly Sp1 and Sp3.