McIlhinney R A, Le Bourdellès B, Molnár E, Tricaud N, Streit P, Whiting P J
Medical Research Council, Anatomical Neuropharmacology Unit, Oxford, UK.
Neuropharmacology. 1998 Oct-Nov;37(10-11):1355-67. doi: 10.1016/s0028-3908(98)00121-x.
The intracellular trafficking, assembly, and cell surface targeting of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A has been studied using both transiently and permanently transfected mammalian cell lines. The expression of either NR1a or NR2A alone does not result in significant cell surface expression of either subunit as determined by cell surface biotinylation and immunofluorescence staining. When NR1a is expressed alone large intracellular accumulations of the subunit are formed which do not co-localize with the golgi apparatus markers protein p58 and wheat germ agglutinin, but do co-localize with the endoplasmic reticulum marker calreticulin. Co-expression of NR1a and NR2A results in a reduction of these intracellular accumulations and the appearance of both subunits on the cell surface. Immunoprecipitation of NR1a from in vitro translated subunit proteins showed that NR2A could only be immunoprecipitated with NR1a when both subunits were co-synthesized in the presence of microsomes. When cells expressing NR1a and NR2A were incubated with [35S]methionine in the presence of Brefeldin-A, a drug which prevents protein transport from the endoplasmic reticulum, NR2A could be immunoprecipitated by an antiserum specific for NR1a. Together these results suggest that the NMDA receptor subunits are assembled in the endoplasmic reticulum and that co-synthesis of the subunits is necessary for their association and their successful cell surface targeting.
利用瞬时转染和永久转染的哺乳动物细胞系，对人 N-甲基-D-天冬氨酸受体亚基 NR1a 和 NR2A 的细胞内运输、组装及细胞表面靶向进行了研究。通过细胞表面生物素化和免疫荧光染色测定，单独表达 NR1a 或 NR2A 均不会导致任一亚基在细胞表面的显著表达。单独表达 NR1a 时，会形成该亚基在细胞内的大量聚集，这些聚集物不与高尔基体标志物蛋白 p58 和麦胚凝集素共定位，但与内质网标志物钙网蛋白共定位。NR1a 和 NR2A 共表达会导致这些细胞内聚集物减少，且两个亚基均出现在细胞表面。对体外翻译的亚基蛋白进行 NR1a 的免疫沉淀显示，只有当两个亚基在微粒体存在的情况下共同合成时，NR2A 才能与 NR1a 一起被免疫沉淀。当用布雷菲德菌素 A（一种阻止蛋白质从内质网转运的药物）处理表达 NR1a 和 NR2A 的细胞，并在[35S]甲硫氨酸存在的情况下进行孵育时，NR2A 可被 NR1a 特异性抗血清免疫沉淀。这些结果共同表明，NMDA 受体亚基在内质网中组装，亚基的共同合成对于它们的结合以及成功靶向细胞表面是必要的。
