Vol. 52, Issue 6, 1019-1026, 1997

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 _1B-Adrenergic Receptor Gene in DDT₁MF-2 Cells

Bin Gao, Jianping Chen, Carl Johnson, and George Kunos

Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

cAMP markedly increases _1B adrenergic receptor (_1B-AR) expression in FRTL-5 and PC C13 rat thyroid cells, DDT₁MF-2 smooth muscle cells, primary rat hepatocytes, and K9 rat liver cells. Here, we used DDT₁MF-2 cells to evaluate further the mechanisms by which cAMP stimulates _1B-AR expression. Receptor binding assays, Northern blotting, and nuclear run-on analyses demonstrated that forskolin (1 µM) in the presence of isobutylmethylxanthine (0.25 mM) increased _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. Dibutyryl cAMP (1 mM) plus isobutylmethylxanthine (0.25 mM) also enhanced _1B-AR density by 2.7 ± 0.1-fold over control. Further experiments demonstrated that the induction of _1B-AR by forskolin requires new protein synthesis and is protein kinase A dependent. In DDT₁MF-2 cells transfected with _1B-AR gene P2 promoter/CAT constructs, both forskolin and dibutyryl cAMP significantly increased P2 promoter activity. The P2 promoter region of the rat _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 elements alone led to a decrease in both basal and cAMP-induced P2 promoter 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 shift and supershift assays using liver nuclear extracts from either rat liver or DDT₁MF-2 cells demonstrated that the CRE in the _1B-AR gene bound CRE binding protein. These data indicate that both the CRE and the AP2 element in the P2 promoter contribute to basal as well as cAMP-induced transcription of the _1B-AR gene in DDT₁MF-2 cells.