CLONING AND IDENTIFICATION THROMBIN RECEPTOR GENE

Thrombin, via activation of vascular endothelial and smooth muscle cel l thrombin receptors, modulates vascular wall healing. To understand t he mechanisms that regulate human thrombin receptor (HTR) expression, we cloned and characterized the HTR gene. The HTR gene consists of Exo n I, which contains the 5'-regulatory region and 85 nucleotides of cod ing sequence; a similar to 15-kb intron; and Exon II, which contains t he remainder of the coding sequence and the entire 3'-untranslated reg ion, Multiple transcription initiation sites were identified by S1 map ping and ribonuclease protection assay. DNA sequence analysis indicate d the presence of two SP-1-AP-2 consensus binding sequences, near or w ithin the transcription initiation sites, and consensus binding sequen ces for numerous regulatory proteins that potentially modulate HTR exp ression. Functional analysis of the HTR promoter was performed by tran sfecting human microvascular endothelial cells with HTR promoter regio n-luciferase constructs. The highest level of expression was obtained with a 0.7-kb promoter sequence and was progressively less with fragme nts of 0.54, 1.16, 1.6, and similar to 3.2 kb. The data presented in t his report provide a foundation for further characterization of the HT R gene and the mechanisms that regulate its expression within the bloo d vessel wall.