SP1-MEDIATED TRANSCRIPTIONAL ACTIVATION FROM THE DOMINANT PROMOTER OFTHE RAT ALPHA(1B) ADRENERGIC-RECEPTOR GENE IN DDT1MF-2 CELLS

In the rat liver, NF1 and CP1 bind to the major P2 promoter of the alp ha(1B) adrenergic receptor gene to generate footprint II. Here we show that, in DDT1MF-2 smooth muscle cells, the major protein bound to foo tprint II is not NFI but Sp1, which binds to the 5'-portion of the foo tprint II sequence (footprint IIb), Mutational analyses demonstrate th at the CCCGCG sequence in footprint IIb is critical for Sp1 binding an d P2 promoter activity. a second GC box in the P2 promoter also binds the Sp1 protein and contributes to the P2 promoter activity. Gel shift assays indicate that footprint II can bind Sp1, NF1, and CP1, and tha t the binding of these 3 proteins is mutually exclusive. This is also indicated by the results of functional cotransfection experiments, wha re transient overexpression of NF1 and Sp1 together caused a similar i ncrease in the activity of a P2/CAT reporter construct as overexpressi on of either Sp1 or NF1 alone, indicating lack of additivity. The pref erential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF 1 in Liver appears to be due to low levels of NF1 expression in DDT1MF -2 cells and low levels of Sp1 in liver. These observations suggest. t hat NF1 and Sp1 are the major transcription factors involved in contro lling the P2 promoter in liver versus DDT1MF-2 cells, respectively, wh ich may be one of the mechanisms responsible for the complex tissue-sp ecific regulation of the expression of the alpha(1B) adrenergic recept or gene.