BOTH THE CYCLIC-AMP RESPONSE ELEMENT AND THE ACTIVATOR PROTEIN-2 BINDING-SITE MEDIATE BASAL AND CYCLIC AMP-INDUCED TRANSCRIPTION FROM THE DOMINANT PROMOTER OF THE RAT ALPHA(1B)-ADRENERGIC RECEPTOR GENE IN DDT1MF-2 CELLS

cAMP markedly increases alpha(1B) adrenergic receptor (alpha(1B)-AR) e xpression in FRTL-5 and PC C13 rat thyroid cells, DDT1MF-2 smooth musc le cells, primary rat hepatocytes, and K9 rat liver cells, Here, we us ed DDT1MF-2 cells to evaluate further the mechanisms by which cAMP sti mulates alpha(1B)-AR expression. Receptor binding assays, Northern blo tting, and nuclear run-on analyses demonstrated that forskolin (1 mu M ) in the presence of isobutylmethylxanthine (0.25 mM) increased alpha( 1B)-AR numbers, mRNA level, and gene transcription rate by 2.3 +/- 0.2 -, 2.5 +/- 0.3-, and 3.5 +/- 0.2-fold over control, respectively. Dibu tyryl cAMP (1 mM) plus isobutylmethylxanthine (0.25 mM) also enhanced alpha(1B)-AR density by 2.7 +/- 0.1-fold over control. Further experim ents demonstrated that the induction of alpha(1B)-AR by forskolin requ ires new protein synthesis and is protein kinase A dependent. In DDT1M F-2 cells transfected with alpha(1B)-AR gene P2 promoter/CAT construct s, both forskolin and dibutyryl cAMP significantly increased P2 promot er activity. The P2 promoter region of the rat alpha(1B)-AR gene (-813 to -432) contains a cAMP response element (CRE) (-444 to -437) and an AP2 binding site (-647 to -638). Mutations in either one of these ele ments alone led to a decrease in both basal and cAMP-induced P2 promot er activity, Mutations in both elements caused a further inhibition of basal transcription and a complete block of cAMP-induced P2 promoter activity. Direct binding of purified activator protein 2 (AP2) to the AP2 element in the P2 promoter was reported previously, Gel mobility s hift and supershift assays using liver nuclear extracts from either ra t liver or DDT1MF-2 cells demonstrated that the CRE in the alpha(1B)-A R gene bound CRE binding protein. These data indicate that both the CR E and the AP2 element in the P2 promoter contribute to basal as well a s cAMP-induced transcription of the alpha(1B)-AR gene in DDT1MF-2 cell s.