Abstract
The 5-hydroxytryptamine type 2 receptor gene is transcriptionally induced by 5-HT-mediated activation of the 5-HT2 receptor in rat myometrial smooth muscle cells. We recently cloned the promoter of the rat 5-HT2 receptor gene and showed that a 1.4-kilobase promoter construct transfected 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 activity. Deletions from base pair (bp) -1314 to bp -184 (with respect to the major transcriptional start (site) resulted in no changes in constitutive or 5-HT-dependent transcriptional activity. A substantial loss of serotonin-dependent transcriptional activation was observed with a promoter construct from which the bp -184 to -108 sequence was deleted. A sequence [termed the serotonin-1 (S1) element], 5'-AGGTTnnnnnnnAACCT-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 this site, as well as an initiator element, appear to be important. Deletion 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 elements appear to be involved in serotonin-dependent induction of promoter activity. Analysis of the promoter elements that direct constitutive (i.e., serotonin-independent) activity revealed the involvement of a different region. Deletions from bp -1314 to bp -75 resulted in only minor increases in basal promoter activity. Deletion to bp -50 resulted in a 2.5-fold increase in basal promoter activity, whereas deletion 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.
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