N-glycosylation of the human neuropeptide QRFP receptor (QRFPR) is essential for ligand binding and receptor activation
N-glycosylation of the human neuropeptide QRFP receptor (QRFPR) is essential for ligand binding and receptor activation
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人类神经肽 QRFP 受体 (QRFPR) 的 N-糖基化对于配体结合和受体激活至关重要
DOI：
10.1111/jnc.15337
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发表时间：
2021
影响因子：
4.7
通讯作者：
Naiming Zhou
中科院分区：
文献类型：
--
作者：
Weiwei Wang;Yanan Tian;Xiaoliu Shi;Qiang Ma;Yue Xu;Gangjie Yang;Wen Yi;Ying Shi;Naiming Zhou
新鉴定的焦谷氨酰化RF酰胺肽（QRFP）信号传导系统已被证明与调节多种生理过程有关。G蛋白偶联受体（GPCR）优先在细胞外结构域上被N-糖基化。人QRFP受体QRFPR（GPR 103）具有三个N-糖基化共有位点，两个位于N-末端结构域（N5和N19），一个位于第一个细胞外环（ECL 1）（N106）;然而，迄今为止，它们在QRFPR表达和信号传导中的作用尚未确定。在这里，我们结合突变体与谷氨酰胺取代的关键天冬酰胺的共识网站与糖苷酶PNGase F和N-糖基化抑制剂衣霉素，研究N-糖基化在QRFPR细胞表面的表达和信号的调节的效果。用定点突变进行的蛋白质印迹分析显示，N-末端N19和ECL 1中的N106处的两个天冬酰胺，而不是N-末端的N5处的两个天冬酰胺，作为N-糖基化的位点。用PNGase F和衣霉素处理导致两种蛋白质种类减少，约43 kDa和约43 kDa。85 kDa大小，2-4 kDa。用共聚焦显微镜和定量ELISA分析表明，QRFPR的N-糖基化不是靶向细胞膜所必需的。然而，进一步的结合测定和功能测定表明，N-糖基化序列子的去除或用衣霉素处理导致受体与QRFP 26和下游信号传导的相互作用的显著损害。因此，我们的研究结果表明，对于人QRFP受体（QRFPR），N-糖基化对细胞表面表达并不重要，但却是配体结合和受体活化的先决条件。
The newly identified pyroglutamylated RFamide peptide (QRFP) signaling system has been shown to be implicated in regulating a variety of physiological processes. G-protein-coupled receptors (GPCRs) are preferentially N-glycosylated on extracellular domains. The human QRFP receptor QRFPR (GPR103) possesses three N-glycosylation consensus sites, two located on the N-terminal domain (N5 and N19) and one on the first extracellular loop (ECL1) (N106); however, to date, their role in QRFPR expression and signaling has not been established. Here, we combined mutants with glutamine substitution of the critical asparagines of the consensus sites with glycosidase PNGase F and N-glycosylation inhibitor tunicamycin to study the effect of N-glycosylation in the regulation of QRFPR cell surface expression and signaling. Western blot analysis performed with site-directed mutagenesis revealed that two asparagines at N19 in the N-terminus and N106 in ECL1, but not N5 in the N-terminus, served as sites for N-glycosylation. Treatment with PNGase F and tunicamycin resulted in a reduction in both two-protein species, ~43 kDa and.~85 kDa in size, by 2–4 kDa. Analysis with confocal microscopy and quantitative ELISA showed that N-glycosylation of QRFPR is not essentially required for targeting the cell membrane. However, further binding assay and functional assays demonstrated that removal of N-glycosylation sequons or treatment with tunicamycin led to significant impairments in the interaction of receptor with QRFP26 and downstream signaling. Thus, our findings suggest that for the human QRFP receptor (QRFPR), N-glycosylation is not important for cell surface expression but is a pre-requisite for ligand binding and receptor activation.