DETERMINATION OF TRANSCRIPTION INITIATION SITES AND PROMOTER ACTIVITYOF THE HUMAN 5-HT2A RECEPTOR GENE

The regulation of 5-HT2A receptor (5-HT(2A)R) expression has been impl icated in a variety of pathological processes. Previous data addressin g the regulation of this receptor are extremely complicated and contro versial. In order to understand the mechanisms of regulation of this r eceptor, we have identified the promoter region of the human 5-HT(2A)R gene. Anchored PCR has mapped a cluster of transcription initiation s ites at nucleotides -1157, -1137, -1127 (numbered sequentially as site s 1, 2 and 3). An additional initiation site (site 4) was detected at -496, 631 bp downstream of site 3. Promoter activity was defined by tr ansfection studies. Several 5' flanking fragments linked to the human growth hormone reporter gene were transfected into two human cell line s, SHSY-5Y (neuroblastoma) and HeLa (cervix carcinoma) which express 5 -HT(2A)R. A 0.74 kb HaeIII/PvuII fragment, which encompasses the initi ation sites 1 to 3 and 5' of the downstream initiation site 4, exhibit ed significant promoter activity in both cell lines. Inclusion of addi tional sequences upstream (the 1.6 kb PvuII/PvuII fragment) had little effect on the promoter activity, but the extension of the 0.74 kb fra gment downstream to include a 0.45 kb PvuII/SmaI fragment drastically decreased the promoter activity. These results suggest that the promot er activity for human 5-HT(2A)R gene resides in the 0.74 kb HaeIII/Pvu II fragment and the 0.45 kb PvuII/SmaI fragment may contain a silencer for the gene expression.