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 DDT 1 MF-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 Received April 21, 1997; Accepted September 4, 1997 SUMMARY cAMP markedly increases  1B adrenergic receptor ( 1B -AR) expression in FRTL-5 and PC C13 rat thyroid cells, DDT 1 MF-2 smooth muscle cells, primary rat hepatocytes, and K9 rat liver cells. Here, we used DDT 1 MF-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 M) 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 en- hanced  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 DDT 1 MF-2 cells transfected with  1B -AR gene P2 promoter/CAT constructs, both forskolin and dibutyryl cAMP significantly increased P2 promoter activity. The P2 pro- moter 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 super- shift assays using liver nuclear extracts from either rat liver or DDT 1 MF-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 DDT 1 MF-2 cells. The  1B -AR is a G-protein-coupled receptor that plays a key role in a variety of physiological processes, such as car- diac and smooth muscle contractility, contraction of the spleen, liver glycogenolysis, melatonin secretion in the pineal gland, and cell proliferation (1–3). The expression of the  1B -AR gene is regulated by hormonal and developmental factors in a complex tissue-specific manner (4). To under- stand the molecular mechanism for such complex regulation, we cloned and characterized the rat  1B -AR gene (5– 8). The gene is composed of two exons and a single large intron of 16 kb. Primer extension and reverse transcriptase-PCR studies using poly(A) + RNA prepared from rat liver identi- fied three tsp, located between -54 and -57 bp (tsp1) and at -443 bp (tsp2) and a cluster between -1035 and -1340 bp (tsp3) upstream from the translation start codon. Northern blot analyses of  1B -AR mRNA have documented three mRNA species that are 3.3, 2.7, and 2.3 kb. The 3.3-kb species is preferentially expressed in rat liver (9, 10), whereas the 2.7-kb species is dominant and widely expressed in many tissues and cells, including rat liver (9, 10). The low-abundance 2.3-kb species is difficult to detect and has been reported only in rat liver (6) and rat medullary thyroid carcinoma 623 cells (11). A similar pattern of three  1B -AR mRNA species was detected in DDT 1 MF-2 cells (12). The 3.3-, 2.7-, and 2.3-kb mRNAs of the  1B -AR gene in rat liver are likely transcribed from tsp3, tsp2, and tsp1, directed by three distinct promoters: the distal promoter (P3, -1363 to -1107), middle promoter (P2, -813 to -432), and proximal promoter (P1, -127 to -49), respectively (6). However, Ka- This work was supported in part by Grant IN-105U from the American Cancer Society and Grant J-379 from the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust. ABBREVIATIONS: AR, adrenergic receptor; CRE, cAMP-response element; AP2, activator protein 2; tsp, transcription start point(s); PCR, polymerase chain reaction; DMSA, DNA mobility shift assay; CAT, chloramphenicol acetyltransferase; CREB, cAMP-response element binding protein; ATF-1, activating transcription factor 1; IBMX, isobutylmethylxanthine; Bt2cAMP, dibutyryl cAMP; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; bp, base pair(s); kb, kilobase pair(s). 0026-895X/97/061019-08$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 52:1019 –1026 (1997). 1019 at ASPET Journals on July 18, 2018 molpharm.aspetjournals.org Downloaded from