Punnonen E L, Wilke T, von Figura K, Hille-Rehfeld A
Universität Göttingen, Institut für Biochemie II, Germany.
Eur J Biochem. 1996 May 1;237(3):809-18. doi: 10.1111/j.1432-1033.1996.0809p.x.
46-kDa mannose-6-phosphate receptor forms homooligomers in cell membranes and in detergent solution. The quaternary structure of detergent-solubilized 46-kDa mannose 6-phosphate receptor is regulated by the presence of ligands, pH and receptor concentration [Waheed, A. & von Figura, K. (1990) Eur. J. Biochem. 193, 47-54). To find out whether the intracellular recycling of 46-kDa mannose 6-phosphate receptor is accompanied by changes in its quaternary structure, we have performed chemical cross-linking in membranes of intact cells. In all conditions tested, the dimer was the predominating form (more than 67% of total 46-kDa mannose 6-phosphate receptor). The amount of trimeric and tetrameric forms varied among cell lines and contributed up to 20% of total endogenous 46-kDa mannose 6-phosphate receptor in human and mouse fibroblasts. Within a given cell line, the ratio of the oligomers was not significantly changed upon elevating endosomal pH by bafilomycin A1, upon changes in receptor occupancy (treatment of cells with tunicamycin or use of mouse fibroblasts deficient in 300-kDa mannose 6-phosphate receptor), nor upon depletion of adaptors from clathrin-coated vesicles of the trans Golgi network by brefeldin A. At the cell surface, where 46-kDa mannose 6-phosphate receptor does not bind ligands, the percentage of dimer was similar to that observed intracellularly. Thus, the oligomeric state of 46-kDa mannose 6-phosphate receptor apparently does not change during recycling as well as binding and dissociation of ligands. In view of the abundance of the dimer of 46-kDa mannose 6-phosphate receptor in situ, our data suggest that it represents the main physiologically active form of the receptor, and therefore present indirect evidence that binding of ligands to 46-kDa mannose 6-phosphate receptor is probably regulated by conformational changes of receptor or ligand rather than by changes in the quaternary structure.
46 kDa甘露糖-6-磷酸受体在细胞膜和去污剂溶液中形成同型寡聚体。去污剂溶解的46 kDa甘露糖6-磷酸受体的四级结构受配体的存在、pH值和受体浓度的调节[瓦希德，A. & 冯·菲古拉，K.（1990年）《欧洲生物化学杂志》193卷，47 - 54页]。为了弄清楚46 kDa甘露糖6-磷酸受体的细胞内再循环是否伴随着其四级结构的变化，我们在完整细胞的膜中进行了化学交联。在所有测试条件下，二聚体是主要形式（占总46 kDa甘露糖6-磷酸受体的67%以上）。三聚体和四聚体形式的数量在不同细胞系中有所不同，在人和小鼠成纤维细胞中占内源性46 kDa甘露糖6-磷酸受体总量的比例高达20%。在给定的细胞系中，通过巴弗洛霉素A1提高内体pH值、受体占有率的变化（用衣霉素处理细胞或使用缺乏300 kDa甘露糖6-磷酸受体的小鼠成纤维细胞），以及通过布雷菲德菌素A从反式高尔基体网络的网格蛋白包被小泡中耗尽衔接蛋白后，寡聚体的比例没有显著变化。在细胞表面（46 kDa甘露糖6-磷酸受体不结合配体），二聚体的百分比与细胞内观察到的相似。因此，46 kDa甘露糖6-磷酸受体的寡聚状态在再循环以及配体的结合和解离过程中显然没有变化。鉴于原位46 kDa甘露糖6-磷酸受体二聚体的丰度，我们的数据表明它代表受体的主要生理活性形式，因此提供了间接证据，表明配体与46 kDa甘露糖6-磷酸受体的结合可能受受体或配体构象变化的调节，而不是受四级结构变化的调节。
