D'Angelo D D, Davis M G, Ali S, Dorn G W
Department of Internal Medicine, University of Cincinnati, Ohio.
J Pharmacol Exp Ther. 1994 Nov;271(2):1034-41.
Pharmacologic and molecular evidence conflicts in regard to the existence of tissue-specific subtypes of thromboxane A2 receptors (TXR). The full length TXR complementary DNA (cDNA) was cloned from a platelet-like cell line. It was expressed and its pharmacology was characterized. Northern analysis of TXR transcripts in multiple tissues showed strong hybridization to K562 chronic myelogenous leukemia messenger RNA. Therefore, a K562 cDNA library was screened and a full-length TXR cDNA (K562TXR) was isolated. K562TXR encodes a protein identical to the previously characterized placenta TXR cDNA, except for a single amino acid substitution (Glu21-->Lys). Similar to thromboxane receptors on K562 cells, K562TXR transiently expressed in HEK 293 cells (K562TXR/293) bound the thromboxane agonist 125I-labeled [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5- heptenoic acid ([125I]BOP) with a Kd of 5.5 +/- 1.1 nM, a Bmax of 289,056 +/- 60,220 sites/cell and a Hill coefficient of -0.94 +/- 0.01 (n = 6). K562TXR/293 cells also demonstrated concentration-dependent increases in intracellular calcium in response to the thromboxane agonist (15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid. In contrast to the single [125I]BOP binding site observed in K562TXR/293, [125I]BOP binding to placental membranes resulted in a Hill coefficient significantly less than unity with a statistically superior two-site model for binding [KdH 0.63 +/- 0.18 nM and KdL of 12.5 +/- 5.0 nM, with Bmaxs of 29 +/- 9 and 212 +/- 41 fmol/mg of protein, respectively (n = 7)].(ABSTRACT TRUNCATED AT 250 WORDS)
关于血栓素A2受体（TXR）组织特异性亚型的存在，药理学和分子学证据存在冲突。从一种血小板样细胞系中克隆出全长TXR互补DNA（cDNA）。对其进行表达并对其药理学特性进行表征。对多个组织中TXR转录本的Northern分析显示，其与K562慢性粒细胞白血病信使核糖核酸有强烈杂交。因此，对K562 cDNA文库进行筛选，分离出一个全长TXR cDNA（K562TXR）。K562TXR编码一种与先前表征的胎盘TXR cDNA相同的蛋白质，只是有一个氨基酸取代（Glu21→Lys）。与K562细胞上的血栓素受体类似，在HEK 293细胞中瞬时表达的K562TXR（K562TXR/293）与血栓素激动剂125I标记的[1S-(1α,2β(5Z),3α-(1E,3S),4α]-7-[3-(3-羟基-4-(对碘苯氧基)-1-丁烯基)-7-氧杂双环-[2.2.1]庚烷-2-基]-5-庚烯酸（[125I]BOP）结合，解离常数（Kd）为5.5±1.1 nM，最大结合容量（Bmax）为289,056±60,220位点/细胞，希尔系数为-0.94±0.01（n = 6）。K562TXR/293细胞对血栓素激动剂（15S-羟基-11α,9α(环氧亚甲基)-前列腺-5Z,13E-二烯酸）的反应也表现出细胞内钙浓度的依赖性增加。与在K562TXR/293中观察到的单一[125I]BOP结合位点相反，[125I]BOP与胎盘膜的结合导致希尔系数显著小于1，且具有统计学上更优的双位点结合模型[高亲和力解离常数（KdH）为0.63±0.18 nM，低亲和力解离常数（KdL）为12.5±5.0 nM，最大结合容量分别为29±9和212±41 fmol/mg蛋白质（n = 7）]。（摘要截断于250字）
