Nguyen L T, Lum H, Tiruppathì C, Malik A B
Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.
Am J Physiol. 1997 Nov;273(5):C1756-63. doi: 10.1152/ajpcell.1997.273.5.C1756.
Thrombin receptor is activated by thrombin-mediated cleavage of the receptor's NH2 terminus between Arg-41 and Ser-42, generating a new NH2 terminus that functions as a "tethered ligand" by binding to sites on the receptor. We prepared antibodies (Abs) directed against specific receptor domains to study the tethered ligand-receptor interactions required for signaling the increase in endothelial permeability to albumin. We used polyclonal Abs directed against the peptide sequences corresponding to the extracellular NH2 terminus [residues 70-99 (AbDD) and 1-160 (AbEE)] and extracellular loops 1 and 2 [residues 161-178 (AbL1) and 244-265 (AbL2)] of the seven-transmembrane thrombin receptor. Receptor activation was determined by measuring changes in cytosolic Ca2+ concentration ([Ca2+]i) in human dermal microvascular endothelial cells (HMEC) loaded with Ca(2+)-sensitive fura 2-acetoxymethyl ester dye. The transendothelial 125I-labeled albumin clearance rate (a measure of endothelial permeability) was determined across the confluent HMEC monolayers. AbEE (300 micrograms/ml), directed against the entire extracellular NH2-terminal extension, inhibited the thrombin-induced increases in [Ca2+]i and the endothelial 125I-albumin clearance rate (> 90% reduction in both responses). AbDD (300 micrograms/ml), directed against a sequence within the NH2-terminal extension, inhibited 70% of the thrombin-induced increase in [Ca2+]i and 60% of the increased 125I-albumin clearance rate. AbL2 (300 micrograms/ml) inhibited these responses by 70 and 80%, respectively. However, AbL1 (300 micrograms/ml) had no effect on either response. We conclude that NH2-terminal extension and loop 2 are critical sites for thrombin receptor activation in endothelial cells and thus lead to increased [Ca2+]i and transendothelial permeability to albumin.
凝血酶受体通过凝血酶介导的受体NH2末端在精氨酸-41和丝氨酸-42之间的裂解而被激活，产生一个新的NH2末端，该末端通过与受体上的位点结合而作为“拴系配体”发挥作用。我们制备了针对特定受体结构域的抗体（Abs），以研究内皮细胞对白蛋白通透性增加信号传导所需的拴系配体-受体相互作用。我们使用了针对与七跨膜凝血酶受体的细胞外NH2末端[残基70-99（AbDD）和1-160（AbEE）]以及细胞外环1和2[残基161-178（AbL1）和244-265（AbL2）]相对应的肽序列的多克隆抗体。通过测量加载有钙敏感的呋喃2-乙酰氧基甲酯染料的人真皮微血管内皮细胞（HMEC）中胞质Ca2+浓度（[Ca2+]i）的变化来确定受体激活。跨汇合的HMEC单层测定跨内皮125I标记的白蛋白清除率（内皮通透性的一种度量）。针对整个细胞外NH2末端延伸的AbEE（300微克/毫升）抑制了凝血酶诱导的[Ca2+]i增加和内皮125I-白蛋白清除率（两种反应均降低>90%）。针对NH2末端延伸内序列的AbDD（300微克/毫升）抑制了70%的凝血酶诱导的[Ca2+]i增加和60%的增加的125I-白蛋白清除率。AbL2（300微克/毫升）分别抑制这些反应70%和80%。然而，AbL1（300微克/毫升）对两种反应均无影响。我们得出结论，NH2末端延伸和环2是内皮细胞中凝血酶受体激活的关键位点，因此导致[Ca2+]i增加和跨内皮对白蛋白的通透性增加。
