Extracellular domain N-glycosylation controls human thrombopoietin receptor cell surface levels.
Extracellular domain N-glycosylation controls human thrombopoietin receptor cell surface levels.
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DOI：
10.3389/fendo.2011.00071
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发表时间：
2011
影响因子：
5.2
通讯作者：
Constantinescu SN
中科院分区：
文献类型：
--
作者：
Albu RI;Constantinescu SN
血小板生成素受体（TpoR）是介导血小板生成素（Tpo）在调节巨核细胞分化、血小板形成和造血干细胞更新中的信号传导功能的I型跨膜蛋白。我们探讨了细胞表面定位和受体功能的四个胞外结构域推定的N-糖基化位点的作用。在四个位点中的任何一个的单个N-糖基化突变体能够获得成熟的N-糖基化模式，但在稳定转导的Ba/F3或Ba/F3-JAK 2细胞系中表现出降低的Tpo依赖性JAK 2-STAT应答。JAK 2促进细胞表面定位和TpoR稳定性的能力需要第一个N-糖基化位点（Asn 117）。相反，第三个N-糖基化位点（Asn 298）降低受体的成熟和稳定性。缺乏三个N-糖基化位点的TpoR突变体在成熟中是有缺陷的，但是可以通过对PNGaseF的敏感性来检测单个剩余位点上的N-糖基化。在所有四个N-糖基化位点缺陷的TpoR突变体在质膜定位中严重受损，并被蛋白酶体降解。N-糖基化受体突变体不会错误折叠，因为一旦在过表达条件下定位于细胞表面，它们就可以结合并响应Tpo。我们的数据表明，细胞外结构域N-糖基化位点调节TpoR的细胞表面定位的组合方式。我们讨论了TpoR N-糖基化位点周围的突变如何可能导致低效的受体运输和疾病。
The thrombopoietin receptor (TpoR) is a type I transmembrane protein that mediates the signaling functions of thrombopoietin (Tpo) in regulating megakaryocyte differentiation, platelet formation, and hematopoietic stem cell renewal. We probed the role of each of the four extracellular domain putative N-glycosylation sites for cell surface localization and function of the receptor. Single N-glycosylation mutants at any of the four sites were able to acquire the mature N-glycosylated pattern, but exhibited a decreased Tpo-dependent JAK2–STAT response in stably transduced Ba/F3 or Ba/F3-JAK2 cell lines. The ability of JAK2 to promote cell surface localization and stability of TpoR required the first N-glycosylation site (Asn117). In contrast, the third N-glycosylation site (Asn298) decreased receptor maturation and stability. TpoR mutants lacking three N-glycosylation sites were defective in maturation, but N-glycosylation on the single remaining site could be detected by sensitivity to PNGaseF. The TpoR mutant defective in all four N-glycosylation sites was severely impaired in plasma membrane localization and was degraded by the proteasome. N-glycosylation receptor mutants are not misfolded as, once localized on the cell surface in overexpression conditions, they can bind and respond to Tpo. Our data indicate that extracellular domain N-glycosylation sites regulate in a combinatorial manner cell surface localization of TpoR. We discuss how mutations around TpoR N-glycosylation sites might contribute to inefficient receptor traffic and disease.