内吞作用
绿色荧光蛋白
转染
分子生物学
内化
受体
HEK 293细胞
缓激肽
化学
细胞生物学
兴奋剂
缓激肽受体
细胞培养
生物
生物化学
基因
遗传学
作者
Dimcho Bachvarov,Sylvain Houle,Magdalena Bachvarova,Johanne Bouthillier,Albert Adam,François Marceau
出处
期刊:PubMed
日期:2001-04-01
卷期号:297 (1): 19-26
被引量:72
摘要
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.
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