Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function.
Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function.
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DOI：
10.1210/mend.9.2.7776966
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发表时间：
1995-02
影响因子：
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通讯作者：
D. Davis;Xuebo Liu;D. Segaloff
中科院分区：
文献类型：
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作者：
D. Davis;Xuebo Liu;D. Segaloff
FSH受体（FSHR）含有一个大的胞外结构域，其中存在三个潜在的N-连接糖基化位点。在哺乳动物细胞中产生并表达仅代表细胞外结构域的截短形式的FSHR。我们表明，这种截短的受体是糖基化的，通过碳水化合物不完全处理的全长受体。这种截短的受体保留在细胞内，以与全长FSHR相当的亲和力结合FSH。因此，尽管FSHR的其他区域可能有助于激素结合，但单独的胞外结构域可以赋予高亲和力结合。上述结果表明，N-连接的FSHR碳水化合物可能以某种方式是FSH结合所必需的。因此，在全长受体的背景下进行进一步的实验，以确定FSHR中糖基化的实际位点以及阐明它们在FSHR功能中的作用。进行定点突变以单独或共同破坏N-连接糖基化的潜在位点。野生型与突变型受体的蛋白质印迹分析表明，在N-连接糖基化的三个潜在位点中，Asn的174和276实际上是糖基化的。结合试验表明，这两个N-连接的FSHR碳水化合物在功能上是冗余的，因为Asn 174或Asn 276处的碳水化合物允许受体在细胞表面上表达并以正常亲和力结合FSH。然而，FSH结合活性没有观察到与非糖基化突变受体，其中两个网站已被集体破坏。类似地，当表达野生型FSHR的细胞用衣霉素处理以防止N-连接的糖基化时，所得的非糖基化FSHR不能结合FSH。相反，当N-连接的碳水化合物从野生型受体中酶促去除时，FSH的正常高亲和力结合得以维持。我们的研究结果表明，虽然N-连接的碳水化合物的FSH受体不需要直接结合激素，碳水化合物在Asn 174或Asn 276是必需的新生受体蛋白质的有效折叠成构象，允许高亲和力结合激素。
The FSH receptor (FSHR) contains a large extracellular domain in which exist three potential sites for N-linked glycosylation. A truncated form of the FSHR representing only the extracellular domain was created and expressed in mammalian cells. We show that this truncated receptor is glycosylated, through the carbohydrates are not as fully processed as those of the full-length receptor. This truncated receptor, which remains intracellular, binds FSH with an affinity comparable to that of the full-length FSHR. Therefore, although other regions of the FSHR may contribute to hormone binding, the extracellular domain alone can confer high affinity binding. The above results suggest that N-linked FSHR carbohydrates may, in some way, be required for FSH binding. Therefore, further experiments, done in the context of the full-length receptor, were performed to determine the actual sites of glycosylation in the FSHR as well as to elucidate their role in the functions of the FSHR. Site-directed mutagenesis was done to individually or collectively disrupt the potential sites for N-linked glycosylation. Western blot analyses of the wild type vs. mutant receptors demonstrate that, of the three potential sites for N-linked glycosylation, Asn's 174 and 276 are actually glycosylated. Binding assays demonstrate that these two N-linked FSHR carbohydrates are redundant in function since carbohydrate at either Asn174 or Asn276 allows the receptor to be expressed on the cell surface and to bind FSH with normal affinity. However, FSH binding activity is not observed with nonglycosylated mutant receptors where both sites have been collectively disrupted. Similarly, when cells expressing the wild type FSHR were treated with tunicamycin to prevent N-linked glycosylation, the resulting nonglycosylated FSHR was not able to bind FSH. In contrast, normal high affinity binding of FSH was maintained when N-linked carbohydrates were enzymatically removed from wild type receptors. Our results demonstrate that while N-linked carbohydrates on the FSH receptor are not required directly for the binding of hormone, a carbohydrate at either Asn174 or Asn276 is required for the efficient folding of the nascent receptor protein into a conformation that allows high affinity binding of hormone.