REGULATION OF RAT 5-HYDROXYTRYPTAMINE TYPE-2 RECEPTOR GENE ACTIVITY -IDENTIFICATION OF CIS-ELEMENTS THAT MEDIATE BASAL AND 5-HYDROXYTRYPTAMINE-DEPENDENT GENE ACTIVATION

The 5-hydroxytryptamine type 2 receptor gene is transcriptionally indu ced by 5-HT-mediated activation of the 5-HT2 receptor in rat myometria l smooth muscle cells. We recently cloned the promoter of the rat 5-HT , receptor gene and showed that a 1.4-kilobase promoter construct tran sfected into myometrial smooth muscle cells displays both constitutive and serotonin-dependent promoter activity, We have examined a series of deletional mutants of this promoter for their transcriptional activ ity, Deletions from base pair (bp) -1314 to bp -184 (with respect to t he major transcriptional start site) resulted in no changes in constit utive or 5-HT-dependent transcriptional activity. A substantial loss o f serotonin-dependent transcriptional activation was observed with a p romoter construct from which the bp -184 to -108 sequence was deleted. A sequence [termed the serotonin-1 (S1) element], 5'-AGGTTnnnnnnnAACC T-3' (where n represents any deoxynucleotide), containing a novel dyad repeat is contained within this region. In addition to the S1 element , two simian virus 40 promoter factor 1 (SP-1) sites contiguous to thi s site, as well as an initiator element, appear to be important. Delet ion of both the S1 and SP-1 sites resulted in an almost total loss of activity. Myometrial smooth muscle cells contain nuclear proteins that interact specifically with the S1 and SP-1 elements. Thus, multiple e lements appear to be involved in serotonin-dependent induction of prom oter activity. Analysis of the promoter elements that direct constitut ive (i.e., serotonin-independent) activity revealed the involvement of a different region, Deletions from bp -1314 to bp -75 resulted in onl y minor increases in basal promoter activity. Deletion to bp -50 resul ted in a 2.5-fold increase in basal promoter activity, whereas deletio n to bp -25 resulted in a 5-fold increase in promoter activity, These results suggest that the basal promoter unit includes bp -25 to +1 and that upstream sequences act to repress basal promoter activity.