Thyrotropin-releasing hormone receptor processing: Role of ubiquitination and proteasomal degradation
Thyrotropin-releasing hormone receptor processing: Role of ubiquitination and proteasomal degradation
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DOI：
10.1210/me.2003-0073
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发表时间：
2003-09-01
影响因子：
--
通讯作者：
Hinkle, PM
中科院分区：
文献类型：
--
作者：
Cook, LB;Zhu, CC;Hinkle, PM
这些研究旨在表征G蛋白偶联TRH受体（TRHR）的泛素化。在中国仓鼠卵巢或垂体GHFT细胞中，TRHRs和泛素与受体或泛素抗体共沉淀。MG-132对蛋白酶体的抑制导致总trhr的积累和少量细胞质受体的出现。MG-132引起新合成受体的增加，通过显微镜使用TRHR偶联Timer检测到，Timer是一种自发的随时间变化的DsRed。错误折叠的trhr尤其被大量泛素化。这些结果表明，蛋白酶体在受体合成后早期参与了TRHR质量控制。在正常情况下，大多数泛素化的trhr被小麦胚芽凝集素吸收，表明它们在高尔基体中经历了复杂的糖基化。当细胞用tunicamycin处理以阻断糖基化时，可以检测到泛素化物种的阶梯。用放射性配体或抗体选择性标记的细胞表面受体没有检测到泛素修饰。为了确定泛素化是否在trh诱导的受体内吞作用中起作用，我们在Ts20细胞中表达了该受体，Ts20细胞具有温度敏感的泛素途径。在允许和非允许温度下，TRH诱导了显著的钙反应和快速而广泛的受体内化，这表明受体或任何其他蛋白质的配体依赖泛素化对于TRHR信号传导或内化都不是必需的。这些结果表明泛素修饰靶向错误折叠的受体进行降解，并提示泛素化在受体运输中的可能作用。
These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.