Identification of the N-linked glycosylation sites of the human relaxin receptor and effect of glycosylation on receptor function.
Identification of the N-linked glycosylation sites of the human relaxin receptor and effect of glycosylation on receptor function.
复制标题
人松弛素受体 N 连接糖基化位点的鉴定以及糖基化对受体功能的影响。
DOI：
10.1021/bi800535b
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
R. Bathgate
中科院分区：
文献类型：
--
作者：
Y. Yan;D. Scott;T. Wilkinson;J. Ji;G. Tregear;R. Bathgate
松弛素受体RXFP 1是富含亮氨酸重复序列的G蛋白偶联受体（LGR）家族的成员。这些受体的特征在于含有富含亮氨酸的重复序列的大的细胞外胞外域，所述富含亮氨酸的重复序列含有主要配体结合位点。RXFP 1在胞外域的Asn-14、Asn-105、Asn-242、Asn-250、Asn-303和Asn-346位置含有6个推定的Asn连接的糖基化位点，这些位点在物种间高度保守。N-连接糖基化是G蛋白偶联受体最常见的翻译后修饰，尽管其在调节受体功能中的作用不同。我们在此研究了RXFP 1的实际N-连接糖基化状态以及这些翻译后修饰的功能性分支。利用定点诱变产生FLAG标记的人RXFP 1的单或多糖基化位点突变体，然后在HEK-293 T细胞中瞬时表达。通过免疫沉淀和蛋白质印迹分析糖基化状态，并通过抗FLAG ELISA、（33）P-H2松弛素竞争结合和cAMP活性测量分析受体功能。在HEK-293 T细胞中利用了RXFP 1的所有潜在N-糖基化位点，重要的是，在单个或双位点和三位点的组合处破坏糖基化对松弛素结合几乎没有影响。然而，细胞表面表达和cAMP信号传导需要糖基化位点的组合。特别是，RXFP 1的Asn-303处的N-糖基化是最佳细胞内cAMP信号传导所必需的。因此，与其他LGR家族成员的情况一样，N-糖基化对于受体转运至细胞表面是必不可少的。此外，糖基化可能对于G蛋白偶联和随后的cAMP信号传导所需的构象变化也是必需的。
The relaxin receptor, RXFP1, is a member of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family. These receptors are characterized by a large extracellular ectodomain containing leucine-rich repeats which contain the primary ligand binding site. RXFP1 contains six putative Asn-linked glycosylation sites in the ectodomain at positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346, which are highly conserved across species. N-Linked glycosylation is the most common post-translational modification of G-protein-coupled receptors, although its role in modulating receptor function differs. We herein investigate the actual N-linked glycosylation status of RXFP1 and the functional ramifications of these post-translational modifications. Site-directed mutagenesis was utilized to generate single- or multiple-glycosylation site mutants of FLAG-tagged human RXFP1 which were then transiently expressed in HEK-293T cells. Glycosylation status was analyzed by immunoprecipitation and Western blot and receptor function analyzed with an anti-FLAG ELISA, (33)P-H2 relaxin competition binding, and cAMP activity measurement. All of the potential N-glycosylation sites of RXFP1 were utilized in HEK-293T cells, and importantly, disruption of glycosylation at individual or combinations of double and triple sites had little effect on relaxin binding. However, combinations of glycosylation sites were required for cell surface expression and cAMP signaling. In particular, N-glycosylation at Asn-303 of RXFP1 was required for optimal intracellular cAMP signaling. Hence, as is the case for other LGR family members, N-glycosylation is essential for the transport of the receptor to the cell surface. Additionally, it is likely that glycosylation is also essential for the conformational changes required for G-protein coupling and subsequent cAMP signaling.