Bachvarov D R, Houle S, Bachvarova M, Bouthillier J, Adam A, Marceau F
Centre Hospitalier Universitaire de Québec, Centre de recherche, Pavillon l'Hôtel-Dieu de Québec, 11 Côte-du-Palais, Québec, Canada G1R 2J6.
J Pharmacol Exp Ther. 2001 Apr;297(1):19-26.
Agonist-induced endocytosis and/or down-regulation have been evaluated using green fluorescent protein (GFP) conjugates of the rabbit bradykinin (BK) B2 receptor (B2R). COS-1 cells transiently transfected with vectors coding for either of two rabbit B2R fluorescent variants, B2R-GFP and B2R-GFP DeltaS/T (with previously identified Ser/Thr phosphorylation sites in the C-terminal tail mutated to Ala), exhibited specific and saturable binding (K(D) in the lower nM range). The acute addition of BK (10-100 nM) to HEK 293 cells stably expressing B2R-GFP in the presence of cycloheximide was rapidly followed by translocation of the surface receptors into the cells, with essentially complete recycling of the surface receptors in 1 to 3 h (confocal microscopy, cell fractionation). Adding captopril to inhibit angiotensin I-converting enzyme activity increased the half-life of BK in the culture medium (enzyme immunoassay) and, accordingly, promoted B2R-GFP internalization for at least 3 h. However, agonist-induced down-regulation was not observed under conditions optimal for endocytosis (microscopy, immunoblot using anti-GFP antibodies). In contrast, B2R-GFP was partially degraded following a short treatment of cells with trypsin. B2R-GFP internalized following agonist treatment was colocalized with fluorescent transferrin, supporting translocation of the receptor to recycling endosomes. B2R-GFP DeltaS/T failed to translocate into the cells following treatment with BK, but exhibited at baseline an altered subcellular distribution relative to B2R-GFP. The agonist BK promotes B(2)R receptor endocytosis followed by recycling to the cell surface, but does not promote receptor down-regulation in the heterologous system that we used here. Digestion initiated by extracellular proteases may be involved in pathological B2R down-regulation, as suggested by the simulation involving trypsin.
使用兔缓激肽（BK）B2受体（B2R）的绿色荧光蛋白（GFP）偶联物评估了激动剂诱导的内吞作用和/或下调。用编码两种兔B2R荧光变体（B2R-GFP和B2R-GFP DeltaS/T，C末端尾巴中先前鉴定的Ser/Thr磷酸化位点突变为Ala）之一的载体瞬时转染的COS-1细胞表现出特异性和可饱和结合（K(D)在较低的纳摩尔范围内）。在环己酰亚胺存在下，向稳定表达B2R-GFP的HEK 293细胞急性添加BK（10-100 nM）后，表面受体迅速转运到细胞内，在1至3小时内表面受体基本完全回收（共聚焦显微镜、细胞分级分离）。添加卡托普利抑制血管紧张素I转换酶活性可增加培养基中BK的半衰期（酶免疫测定），因此促进B2R-GFP内化至少3小时。然而，在内吞作用的最佳条件下未观察到激动剂诱导的下调（显微镜检查、使用抗GFP抗体的免疫印迹）。相反，用胰蛋白酶短暂处理细胞后，B2R-GFP会部分降解。激动剂处理后内化的B2R-GFP与荧光转铁蛋白共定位，支持受体转运至回收内体。用BK处理后，B2R-GFP DeltaS/T未能转运到细胞内，但相对于B2R-GFP，其在基线时亚细胞分布发生了改变。激动剂BK促进B(2)R受体内吞，随后再循环至细胞表面，但在我们这里使用的异源系统中不促进受体下调。如涉及胰蛋白酶的模拟所示，细胞外蛋白酶引发的消化可能参与病理性B2R下调。
