ISOLATION AND CHARACTERIZATION OF THE RAT 5-HYDROXYTRYPTAMINE TYPE-2 RECEPTOR PROMOTER - CONSTITUTIVE AND INDUCIBLE ACTIVITY IN MYOMETRIAL SMOOTH-MUSCLE CELLS

Previous studies from this laboratory have demonstrated that the 5-hyd roxytryptamine (5-HT2) receptor subtype is transcriptionally regulated by 5-HT (serotonin) itself in rat myometrial smooth muscle cells. To better understand this transcriptional regulation, we have isolated an d characterized the 5'-flanking region of the 5-HT2 receptor gene. Scr eening of a rat genomic library was accomplished using 5'-directed fra gments of 5-HT2 cDNA, and a 5.2-kilobase fragment was isolated. Sequen cing demonstrated that the fragment overlapped the 5'-end of the 5-HT2 cDNA by 226 base pairs. Primer extension and RNase protection analyse s indicated that three transcriptional start sites, which are common t o both rat brain and myometrium, appear to exist and that the 5'-untra nslated region of the 5-HT2 receptor cDNA is 1120 base pairs long. Nei ther classical TATA boxes nor CCAAT sequences were found upstream of a ny of the start sites identified. Upstream of the dominant start site, however, an initiator consensus sequence, two GC boxes (SP-1 binding sites), and several AP-2 binding sites were identified. Based on this information, a 1.4-kilobase fragment beginning 64 base pairs downstrea m from the dominant start site was constructed by polymerase chain rea ction and ligated into a pCAT vector. Transient transfection of this c onstruct into rat myometrial smooth muscle cells displayed both consti tutive and seretonin-induced promoter activity. Serotonin-inducible ac tivity was abolished by a selective 5-HT2 receptor antagonist; however , antagonists selective for other 5-HT receptor subtypes were without effect. Conversely, a selective 5-HT2 receptor agonist completely subs tituted for serotonin as an inducer. Preliminary deletion experiments indicate that regulation of basal and seretonin-inducible activity lik ely depends upon different cis elements in the 5-HT2 receptor gene pro moter.