Melikian H E, Ramamoorthy S, Tate C G, Blakely R D
Graduate Program in Neuroscience, Emory University, Atlanta, Georgia 30322, USA.
Mol Pharmacol. 1996 Aug;50(2):266-76.
The role of N-glycosylation in the expression, stability, and ligand recognition by the cocaine- and antidepressant-sensitive human norepinephrine transporter (hNET) was assessed in stably and transiently transfected cell lines. The use of hNET-specific antibodies and the membrane-impermeant biotinylating reagent sulfosuccinimidobiotin establishes that treatment of stably transfected LLC-PK1 cells with tunicamycin depletes surface membranes of mature hNET glycoproteins, which is consistent with a failure of less stable, nonglycosylated subunits to replenish surface compartments. To determine whether N-glycosylation plays a direct role in hNET stability, surface expression, and ligand recognition, we mutated the three hNET canonical N-glycosylation sites (hNETN184, 192, 198Q) and transiently expressed the mutant cDNA in parallel with the parental hNET construct in HeLa and COS cells. hNETN184, 192, 198Q protein exhibited increased electrophoretic mobility (approximately 46 kDa), similar to that of enzymatically N-deglycosylated hNET protein, which confirms the use of canonical sites in the second extracellular loop of the transporter. hNETN184, 192, 198Q protein in HeLa and COS extracts was reduced approximately 50% relative to hNET protein in parallel transfections, demonstrated to arise from a reduction in transporter half-life, which is consistent with the proposed role of N-glycosylation in hNET stability. Both HeLa and COS cells transfected with hNETN184, 192, 198Q exhibit a significantly greater reduction in transport activity than can be accounted for by losses in either total or surface NET protein. Furthermore, sensitivity of catecholamine transport to unlabeled substrate and antagonists was unchanged in the mutant, suggesting that residual nonglycosylated surface hNETs execute a key step in the transport cycle after ligand recognition with reduced efficiency.
在稳定转染和瞬时转染的细胞系中评估了N-糖基化在可卡因和抗抑郁药敏感的人去甲肾上腺素转运体（hNET）的表达、稳定性和配体识别中的作用。使用hNET特异性抗体和膜不透性生物素化试剂磺基琥珀酰亚胺生物素证实，用衣霉素处理稳定转染的LLC-PK1细胞会耗尽成熟hNET糖蛋白的表面膜，这与不稳定的非糖基化亚基无法补充表面区室一致。为了确定N-糖基化是否在hNET稳定性、表面表达和配体识别中起直接作用，我们对hNET的三个典型N-糖基化位点（hNETN184、192、198Q）进行了突变，并在HeLa和COS细胞中与亲本hNET构建体平行瞬时表达突变cDNA。hNETN184、192、198Q蛋白表现出增加的电泳迁移率（约46 kDa），类似于酶促N-去糖基化的hNET蛋白，这证实了转运体第二个细胞外环中典型位点的使用。与平行转染中的hNET蛋白相比，HeLa和COS提取物中的hNETN184、192、198Q蛋白减少了约50%，这表明转运体半衰期缩短，这与N-糖基化在hNET稳定性中的作用一致。用hNETN184、192、198Q转染的HeLa和COS细胞的转运活性降低幅度均显著大于总NET蛋白或表面NET蛋白损失所能解释的程度。此外，突变体中儿茶酚胺转运对未标记底物和拮抗剂的敏感性未改变，这表明残留的非糖基化表面hNETs在配体识别后以较低效率执行转运循环中的关键步骤。
