Mirza H, Yatsula V, Bahou W F
Department of Medicine, State University of New York at Stony Brook, 11794-8151, USA.
J Clin Invest. 1996 Apr 1;97(7):1705-14. doi: 10.1172/JCI118597.
Proteolytically cleaved receptors, typified by the functional thrombin receptor (TR), represent a novel class of receptors that mediate signaling events by functional coupling to G proteins. Northern blot analysis completed with a human proteinase activated receptor-2 (PAR-2) cDNA as probe demonstrated the approximately 3.5kb PAR-2 transcript in total cellular RNA from human umbilical vein endothelial cells (HUVEC). Microspectrofluorimetry using Fura2-loaded HUVEC demonstrated a dose-dependent elevation in intracellular calcium transients ([Ca2+]i) to murine PAR39-44 (SLIGRL, putative neoligand after cleavage), with an approximate EC50 of 30 microM, and evidence for homologous desensitization with complete recovery at 45 min. Xenopus oocytes microinjected with TR cRNA failed to respond to 200 microM PAR39-44, and TR-targeted antisense oligonucleotides specifically abrogated thrombin-induced but not PAR39-44-mediated [Ca2+]i, excluding the possibility that TR/PAR-2 cell-surface coexpression was structurally linked. HUVEC incubated with PAR39-44 demonstrated a dose- and time-dependent mitogenic response similar to that seen with thrombin or TR42-47 (TR-activating peptide, SFLLRN). Preactivation of HUVEC with either PAR39-44 or thrombin resulted in heterologous desensitization to the corresponding agonist, an effect that was mediated primarily by TR internalization as evaluated by immunofluorescence and quantitative ELISA. These results ascribe a previously unrecognized function to the PAR-2 receptor, imply that a natural enzyme agonist may circulate in plasma, and suggest the presence of an additional regulatory mechanism controlling receptor activation events in vascular endothelial cells.
以功能性凝血酶受体（TR）为代表的蛋白水解切割受体，是一类新型受体，通过与G蛋白功能性偶联介导信号转导事件。用人蛋白酶激活受体-2（PAR-2）cDNA作为探针进行的Northern印迹分析表明，在人脐静脉内皮细胞（HUVEC）的总细胞RNA中存在约3.5kb的PAR-2转录本。使用负载Fura2的HUVEC进行的显微分光荧光测定显示，细胞内钙瞬变（[Ca2+]i）对鼠PAR39-44（切割后的假定新配体，SLIGRL）呈剂量依赖性升高，近似EC50为30 microM，并且有同源脱敏的证据，45分钟时完全恢复。显微注射TR cRNA的非洲爪蟾卵母细胞对200 microM PAR39-44无反应，并且TR靶向反义寡核苷酸特异性消除了凝血酶诱导的而非PAR39-44介导的[Ca2+]i，排除了TR/PAR-2细胞表面共表达在结构上相关联的可能性。用PAR39-44孵育的HUVEC表现出与凝血酶或TR42-47（TR激活肽，SFLLRN）相似的剂量和时间依赖性促有丝分裂反应。用PAR39-44或凝血酶预激活HUVEC导致对相应激动剂的异源脱敏，通过免疫荧光和定量ELISA评估，这种效应主要由TR内化介导。这些结果赋予PAR-2受体一种先前未被认识的功能，暗示天然酶激动剂可能在血浆中循环，并提示存在一种额外的调节机制来控制血管内皮细胞中的受体激活事件。
