Abstract
Thrombin, via activation of vascular endothelial and smooth muscle cell thrombin receptors, modulates vascular wall healing. To understand the mechanisms that regulate human thrombin receptor (HTR) expression, we cloned and characterized the HTR gene. The HTR gene consists of Exon I, which contains the 5'-regulatory region and 85 nucleotides of coding sequence; a approximately 15-kb intron; and Exon II, which contains the remainder of the coding sequence and the entire 3'-untranslated region. Multiple transcription initiation sites were identified by S1 mapping and ribonuclease protection assay. DNA sequence analysis indicated the presence of two SP-1-AP-2 consensus binding sequences, near or within the transcription initiation sites, and consensus binding sequences for numerous regulatory proteins that potentially modulate HTR expression. Functional analysis of the HTR promoter was performed by transfecting human microvascular endothelial cells with HTR promoter region-luciferase constructs. The highest level of expression was obtained with a 0.7-kb promoter sequence and was progressively less with fragments of 0.54, 1.16, 1.6, and approximately3.2 kb. The data presented in this report provide a foundation for further characterization of the HTR gene and the mechanisms that regulate its expression within the blood vessel wall.
Highlights
The serine protease, thrombin, mediates several biologic processes that are essential for maintaining vascular integrity
Results were tabulated as counts per second (CPS), and normalized according to alkaline phosphatase (PAP) activity (PAP gene in pSV2Apap) per well. pSV2-luc was used as a positive control and poluc as a negative control for these transfection experiments
Dot hybridization analysis of P1282 and P1287 DNA with a 300-bp human thrombin receptor (HTR) cDNA sequence from the 5Ј end of the cDNA (5Ј-cDNA probe) and a 2-kb intron sequence located at the 3Ј end of the intron indicated that both P1282 and P1287 contained a sequence that hybridized to the Intron probe (Fig. 1C)
Summary
Isolation of Human Thrombin Receptor Genomic DNA Clones—A human genomic DNA bacteriophage P1 library After precipitation with 2.5 volumes of ethanol, DNA probe-poly(Aϩ) RNA mixtures were resuspended in 20 l of S1 hybridization solution containing 40 mM PIPES (pH 6.4), 1 mM EDTA, 0.4 M NaCl, and 80% deionized formamide and incubated at 65 °C for 10 min, at 30 °C overnight for annealing. After transcription for 60 min at 37 °C, the DNA template was digested with 2 units of RNase-free DNase I for 25 min, and full-length antisense RNA probes were recovered from 8 M ureadenatured PAGE gel by elusion buffer (0.5 M ammonium acetate, 1 mM EDTA, 0.2% SDS) at 35 °C overnight. Subcloning and DNA Sequencing Analysis—pT7T318U-HTR/5.5 and pT7T318U-HTR/2.5 were obtained by ligating either a 5.5-kb EcoRI fragment (containing 5Ј sequence, Exon I, and the 5Ј intron-exon junction) or a 2.5-kb EcoRI fragment
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