Neumann D, Wikström L, Watowich S S, Lodish H F
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
J Biol Chem. 1993 Jun 25;268(18):13639-49.
The erythropoietin receptor (EPO-R) is synthesized in transfected Ba/F3 cells as a major 64-kDa endoglycosidase H (Endo H)-sensitive species, with a single N-linked oligosaccharide, and a minor 62-kDa unglycosylated form. Approximately half of the newly made EPO-R is processed to a mature 66-kDa form with a Golgi-processed Endo H-resistant oligosaccharide, of which only a minor fraction is expressed at the cell surface. Both the Endo H-sensitive and the Endo H-resistant forms of the receptor have a half-life of 45-60 min (Yoshimura, A., D'Andrea, A. D., and Lodish, H. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4139-4143). The mature, Endo H-resistant form of the EPO-R appears to be degraded in lysosomes or in other acidic organelles, since receptor degradation is blocked by treatment with NH4Cl, chloroquine, or leupeptin. A fraction of the Endo H-resistant EPO-R molecules is cleaved, generating two fragments of 46 and 39 kDa. The sizes of these fragments and their reactivities with carboxyl-terminal-specific antibodies indicate that the receptor is cleaved at two sites in the exoplasmic domain, 7 kDa apart, and carboxyl-terminal to the N-glycosylation site. Both fragments are membrane anchored and are probably formed in a late or post-Golgi compartment, since their formation is blocked by incubation of cells at 20 degrees C or by incubation with brefeldin A. These membrane-anchored COOH-terminal fragments are probably degraded in lysosomes or in other acidic vesicles as cell fractionation demonstrates that they colocalize with lysosomes, and similar to the intact EPO-R, their degradation is inhibited by NH4Cl. Finally, double labeling immunofluorescence experiments demonstrate that in NH4Cl-treated cells both intact mature EPO-R and the 46- and 39-kDa fragments accumulate in lysosomes and presumably are normally degraded there. The sensitivity of the EPO-R to endoproteolytic cleavages in its exoplasmic domain may relate to its low surface expression and to its extreme metabolic instability.
促红细胞生成素受体（EPO-R）在转染的Ba/F3细胞中合成，主要以一种64 kDa的对内切糖苷酶H（Endo H）敏感的形式存在，带有单个N-连接寡糖，还有一种少量的62 kDa未糖基化形式。新合成的EPO-R中约一半被加工成成熟的66 kDa形式，带有经高尔基体加工的对Endo H有抗性的寡糖，其中只有一小部分在细胞表面表达。受体的Endo H敏感形式和Endo H抗性形式的半衰期均为45 - 60分钟（吉村，A.，丹德利亚，A. D.，和洛迪什，H. F.（1990年）《美国国家科学院院刊》87，4139 - 4143）。EPO-R的成熟、对Endo H有抗性的形式似乎在溶酶体或其他酸性细胞器中被降解，因为用氯化铵、氯喹或亮抑酶肽处理可阻断受体降解。一部分对Endo H有抗性的EPO-R分子被切割，产生46 kDa和39 kDa的两个片段。这些片段的大小及其与羧基末端特异性抗体的反应性表明，受体在外质结构域的两个位点被切割，相距7 kDa，且在N-糖基化位点的羧基末端。两个片段都锚定在膜上，可能在高尔基体晚期或高尔基体后区室形成，因为在20℃孵育细胞或用布雷菲德菌素A孵育会阻断它们的形成。这些锚定在膜上的羧基末端片段可能在溶酶体或其他酸性小泡中被降解，因为细胞分级分离表明它们与溶酶体共定位，并且与完整的EPO-R类似，它们的降解被氯化铵抑制。最后，双重标记免疫荧光实验表明，在氯化铵处理的细胞中，完整的成熟EPO-R以及46 kDa和39 kDa片段都在溶酶体中积累，推测它们通常在那里被降解。EPO-R在外质结构域对内蛋白水解切割的敏感性可能与其低表面表达及其极端的代谢不稳定性有关。
