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(Circulation Research. 1995;77:466-474.)
© 1995 American Heart Association, Inc.

Articles

Characterization of 5' End of Human Thromboxane Receptor Gene

Organizational Analysis and Mapping of Protein Kinase C– Responsive Elements Regulating Expression in Platelets

Drew D. D'Angelo, Michael G. Davis, William A. Houser, Jeremy J. Eubank, Michael E. Ritchie, Gerald W. Dorn, II

From the University of Cincinnati (Ohio) Medical Center.

Correspondence to Gerald W. Dorn II, MD, University of Cincinnati Medical Center, ML 542, 231 Bethesda Ave, Cincinnati, OH 45267-0542.

Abstract Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into plateletlike K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester–responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2–like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.

Key Words: thromboxane receptor • platelets • gene expression • transcriptional regulation • protein kinase C

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