Li F, Baykal D, Horaist C, Yan C N, Carr B N, Rao G N, Runge M S
Cardiology Division and Sealy Center for Molecular Cardiology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
J Biol Chem. 1996 Oct 18;271(42):26320-8. doi: 10.1074/jbc.271.42.26320.
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.
凝血酶通过激活血管内皮细胞和平滑肌细胞的凝血酶受体来调节血管壁愈合。为了解调节人凝血酶受体（HTR）表达的机制，我们克隆并鉴定了HTR基因。HTR基因由外显子I、一个约15kb的内含子和外显子II组成。外显子I包含5'调控区和85个核苷酸的编码序列；外显子II包含其余的编码序列和整个3'非翻译区。通过S1作图和核糖核酸酶保护试验确定了多个转录起始位点。DNA序列分析表明，在转录起始位点附近或内部存在两个SP-1-AP-2共有结合序列，以及许多可能调节HTR表达的调控蛋白的共有结合序列。通过用HTR启动子区域-荧光素酶构建体转染人微血管内皮细胞，对HTR启动子进行了功能分析。用0.7kb的启动子序列获得了最高水平的表达，而用0.54、1.16、1.6和大约3.2kb的片段表达水平逐渐降低。本报告中的数据为进一步鉴定HTR基因及其在血管壁内调节其表达的机制奠定了基础。
