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-hydroxytryptamine (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 and characterized the 5'-flanking region of the 5-HT2 receptor gene. Screening of a rat genomic library was accomplished using 5'-directed fragments of 5-HT2 cDNA, and a 5.2-kilobase fragment was isolated. Sequencing demonstrated that the fragment overlapped the 5'-end of the 5-HT2 cDNA by 226 base pairs. Primer extension and RNase protection analyses indicated that three transcriptional start sites, which are common to both rat brain and myometrium, appear to exist and that the 5'-untranslated region of the 5-HT2 receptor cDNA is 1120 base pairs long. Neither classical TATA boxes nor CCAAT sequences were found upstream of any 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 downstream from the dominant start site was constructed by polymerase chain reaction and ligated into a pCAT vector. Transient transfection of this construct into rat myometrial smooth muscle cells displayed both constitutive and serotonin-induced promoter activity. Serotonin-inducible activity 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 substituted for serotonin as an inducer. Preliminary deletion experiments indicate that regulation of basal and serotonin-inducible activity likely depends upon different cis elements in the 5-HT2 receptor gene promoter.
Department of Pharmacology and Toxicology, Albany Medical College, New York 12208.
Y L
YL
Du
B D
BD
Wilcox
M
M
Teitler
J J
JJ
Jeffrey
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary; D005260:Female; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D009215:Myometrium / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection
D018076:DNA, Complementary; D011985:Receptors, Serotonin