Glycosylation of the human prostacyclin receptor: role in ligand binding and signal transduction.
Glycosylation of the human prostacyclin receptor: role in ligand binding and signal transduction.
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DOI：
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发表时间：
2001-09
影响因子：
3.6
通讯作者：
Z. Zhang;S. Austin;E. Smyth
中科院分区：
文献类型：
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作者：
Z. Zhang;S. Austin;E. Smyth
前列环素是一种有效的血管舒张剂和血小板聚集抑制剂，通过细胞表面 G 蛋白偶联受体 [前列环素 (IP)] 发挥作用。人 (h) IP 包含两个 N 连接糖基化共有位点（N(7) 和 N(78)）。然而，糖基化的作用尚不清楚。通过用Q替换N(7)和/或N(78)生成突变体受体(N(7)-Q(7)、N(78)-Q(78)和N(7)、N(78)-Q(7)、Q(78))。受体糖基化在野生型和N(7)-Q(7)中相似并且被衣霉素抑制。 N(78)-Q(78) 和 N(7),N(78)-Q(7),Q(78) 表现出很少或没有糖基化。每个突变体的膜定位都减少，同时糖基化受损。部分定位于质膜，可以直接检查糖基化对 IP 功能的影响。与 N(7)-Q(7) 的高亲和力结合（K(d) = 21.7 +/- 1.7 nM，n = 4）与野生型受体的高亲和力结合（K(d) = 24.3 +/- 3.6 nM，n = 4）相似，尽管 B(max) 值降低（0.35 +/- 0.03 fmol/mg 蛋白质与 3.34 +/- 0.52 fmol/mg 蛋白质） fmol/mg 蛋白质，n = 4)。与 N(78)-Q(78)（B(max) = 0.27 +/- 0.03 fmol/mg 蛋白质，n = 3；K(d) = 149.1 +/- 11.1，n = 3）和 N(7)、N(78)-Q(7)、Q(78)（无特异性结合）的结合进一步受损。 N(7)-Q(7) 细胞中激动剂诱导的腺苷酸环化酶活化减少，而 N(78)-Q(78) 细胞仅对高浓度伊洛前列素有反应，而 N(7)、N(78)-Q(7)、Q(78) 无反应。仅在野生型中肌醇磷酸盐的产生是明显的。只有野生型和 N(7)-Q(7) 受体经历激动剂诱导的隔离。我们的研究结果表明，与 N(7) 相比，N(78) 的糖基化程度更高。 hIP 的 N 连接糖基化程度对于膜定位、配体结合和信号转导可能很重要。
Prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, acts through a cell-surface G protein-coupled receptor [prostacyclin (IP)]. The human (h) IP contains two consensus sites for N-linked glycosylation (N(7) and N(78)). However, the role of glycosylation is unknown. Mutant receptors (N(7)-Q(7),N(78)-Q(78) and N(7),N(78)-Q(7),Q(78)) were generated by replacing N(7) and/or N(78) with Q's. Receptor glycosylation was similar in the wild-type and N(7)-Q(7) and was inhibited with tunicamycin. N(78)-Q(78) and N(7),N(78)-Q(7),Q(78) demonstrated little or no glycosylation. Membrane localization was reduced for each mutant concomitant with impaired glycosylation. Partial localization to the plasma membrane allowed direct examination of the effect of glycosylation on IP function. High-affinity binding to N(7)-Q(7) was similar (K(d) = 21.7 +/- 1.7 nM, n = 4) to that of the wild-type receptor (K(d) = 24.3 +/- 3.6 nM, n = 4), despite a reduced value for B(max) (0.35 +/- 0.03 fmol/mg of protein versus 3.34 +/- 0.52 fmol/mg of protein, n = 4). Binding to N(78)-Q(78) (B(max) = 0.27 +/- 0.03 fmol/mg of protein, n = 3; K(d) = 149.1 +/- 11.1, n = 3) and N(7),N(78)-Q(7),Q(78) (no specific binding) was further impaired. Agonist-induced adenylyl cyclase activation was reduced in N(7)-Q(7) cells, whereas N(78)-Q(78) cells responded only to high concentrations of iloprost and N(7),N(78)-Q(7),Q(78) were unresponsive. Inositol phosphate generation was evident only with the wild-type. Only the wild-type and N(7)-Q(7) receptors underwent agonist-induced sequestration. Our findings demonstrate greater glycosylation at N(78) compared with N(7). The extent of N-linked glycosylation of hIP may be important for membrane localization, ligand binding, and signal transduction.