CHARACTERIZATION OF 5'-END OF HUMAN THROMBOXANE RECEPTOR GENE - ORGANIZATIONAL ANALYSIS AND MAPPING OF PROTEIN-KINASE C-RESPONSIVE ELEMENTSREGULATING EXPRESSION IN PLATELETS

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxan e receptors are under transcriptional control, we isolated and charact erized human genomic DNA clones containing the 5' flanking region of t he thromboxane receptor gene. The exon-intron structure of the 5' port ion of the thromboxane receptor gene was determined initially by compa ring the nucleotide sequence of the 5' flanking genomic clone with tha t of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human pla cental cDNA. A major transcription initiation site was located in thre e human tissues approximate to 560 bp upstream from the translation in itiation codon and 350 bp upstream from any previously identified tran scription 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 acetyltransferas e (CAT) chimera plasmids into plateletlike K562 cells. Thromboxane rec eptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Funct ional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motif s in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximate to 1.8 kb 5' from the transcription initiation site. These studies are the firs t to determine the structure and organization of the 5' end of the thr omboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via indu ction of an AP-2-like nuclear factor binding to upstream promoter elem ents. These findings strongly suggest that the mechanism for previousl y described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcri ption in platelet-progenitor cells.