Determination of transcription initiation sites and promoter activity of the human 5-HT2A receptor gene.
The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes. Previous data addressing the regulation of this receptor are extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, we have identified the promoter region of the human 5-HT2AR gene. Anchored PCR has mapped a cluster of transcription initiation sites at nucleotides -1157, -1137, -1127 (numbered sequentially as sites 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 transfection studies. Several 5' flanking fragments linked to the human growth hormone reporter gene were transfected into two human cell lines, SHSY-5Y (neuroblastoma) and HeLa (cervix carcinoma) which express 5-HT2AR. A 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites 1 to 3 and 5' of the downstream initiation site 4, exhibited significant promoter activity in both cell lines. Inclusion of additional sequences upstream (the 1.6 kb PvuII/PvuII fragment) had little effect on the promoter activity, but the extension of the 0.74 kb fragment downstream to include a 0.45 kb PvuII/SmaI fragment drastically decreased the promoter activity. These results suggest that the promoter activity for human 5-HT2AR gene resides in the 0.74 kb HaeIII/PvuII fragment and the 0.45 kb PvuII/SmaI fragment may contain a silencer for the gene expression.
Shih
J C
JC
Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles 90033, USA.
Zhu
Q
Q
Chen
K
K
D001483:Base Sequence; D001923:Brain Chemistry / Q000502:physiology; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000502:physiology*
D011985:Receptors, Serotonin