Identification of Three Alternative First Exons and an Intronic Promoter of Human PDE5A Gene Ching-Shwun Lin, 1 Angie Lau, Richard Tu, and Tom F. Lue Knuppe Molecular Urology Laboratory, Department of Urology, University of California, San Francisco, California 94143-1695 Received December 28, 1999 In the accompanying paper we present evidence for the existence of three PDE5A isoforms that differed only in the 5 end of the mRNAs. In this paper we present evidence that the three isoform-specific 5 ends were encoded by three alternative first exons that were arranged in the order of A1–A3–A2. Because the isoform-specific mRNAs could be transcribed from individual promoters, DNA fragments of the two in- tronic regions (A1–A3 and A3–A2) were tested for pos- sible promoter activities. The intron between A1- and A3-specific exons did not exhibit any promoter activi- ties even in smooth muscle cells that expressed the A3 isoform (see accompanying paper). In contrast, the intron between A3- and A2-specific exons had pro- moter activities in PDE5A2-expressing COS-7 and smooth muscle cells. This intronic promoter was bound by transcription factors AP-2 and Sp1, but not by AP-1, as shown by DNase I footprint analysis. How- ever, the sequence bound by AP-2 (5-GGGAAACG- CTCGCGGGAGAGTTGG) is unusual in that it bears little resemblance to the consensus AP-2-binding sequence. © 2000 Academic Press Cyclic GMP mediates most of the nonlytic physiolog- ical effects of nitric oxide (NO) and all of the known physiological effects of natriuretic peptides and guany- lins. For example, it stimulates smooth muscle relax- ation, inhibits platelet aggregation, and initiates vi- sual signal transduction. Tissue cGMP concentrations are determined by guanylyl cyclases that catalyze the formation of cGMP from GTP and by phosphodiester- ases (PDEs) that catalyze the breakdown of cGMP (1). Maintaining cGMP levels through inhibition of a cGMP-specific PDE (PDE5) has proven to be an effec- tive means for enhancing penile smooth muscle relax- ation (2). However, previous reports have demon- strated the widespread distribution of PDE5 mRNA in various tissues (3– 6), raising the possibility that PDE5-inhibition-based medications might have ad- verse systemic side effects. An added complication is the fact that multiple forms of PDE5 exist (3– 6), in- cluding a third isoform that we have recently identified (see accompanying paper). In vitro tests have shown little differences among the three isoforms in cGMP- catalytic activities and in sensitivities to PDE5-specific inhibitors (5). However, it remains possible that the isoforms may possess in vivo functional differences, as suggested by evidence that their tissue distribution patterns differ. For example, we have found that the PDE5A2 isoform was the predominant form in the majority of tissues and cultured cell types, and the PDE5A3 isoform might be expressed specifically in smooth muscles (see accompanying paper). The differences in tissue distribution would suggest that the expression of the three PDE5A isoforms might be differentially regulated in physiological conditions or in response to medications. To investigate this pos- sibility, we took the approach of cloning the genetic elements that control the expression of PDE5 isoforms. The approach was based on the information that the three PDE5A isoform mRNAs differ only in the 5' end and therefore are mostly likely generated by the splic- ing of three alternative first exons. These alternative exons could be transcribed in a single pre-mRNA from a single gene promoter or in multiple pre-mRNAs from multiple gene promoters. In this report we present evidence that the three alternative PDE5A first exons were positioned in the order of A1–A3–A2 and that the A2 exon was preceded by a potent intronic promoter. MATERIALS AND METHODS Cell culture. Monkey fibroblast cell line COS-7 was purchased from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Human aorta smooth muscle cells (AOSMC) were purchased from Clonetics Inc. (San Diego, CA) and grown in smooth muscle growth medium (Clonetics). 1 To whom correspondence should be addressed. Fax: 415-353- 9586. E-mail: cslin@itsa.ucsf.edu. Biochemical and Biophysical Research Communications 268, 596 – 602 (2000) doi:10.1006/bbrc.2000.2186, available online at http://www.idealibrary.com on 596 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.