Couvineau A, Fabre C, Gaudin P, Maoret J J, Laburthe M
Unité de Recherche de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine Xavier-Bichat, Paris, France.
Biochemistry. 1996 Feb 13;35(6):1745-52. doi: 10.1021/bi952022h.
The functional role of N-linked carbohydrates in the human vasoactive intestinal peptide (VIP) 1 receptor was investigated by site-directed mutagenesis (Asn-->Thr) of the four consensus N-glycosylation sites on Asn58, Asn69, Asn100 (N-terminal extracellular domain) and Asn293 (second extracellular loop). Mutated receptors were investigated after transient expression in Cos-7 cells, by ligand binding assay, affinity cross-linking, western blotting, and confocal laser microscopy of epitope-tagged receptor proteins. Mutations of each consensus site revealed that Asn58, Asn69, and Asn100 were occupied by a 9-kDa N-linked carbohydrate whereas Asn293 was not used for glycosylation. Each mutated receptor was expressed (western blot) and delivered at the plasma membrane (confocal microscopy) of Cos-7 cells. They displayed a dissociation constant similar to that of the wild-type receptor, i.e., 0.5-1 nM. In contrast, no VIP binding to Cos-7 cells could be observed with the mutant devoid of consensus N-glycosylation sites due to a strict sequestration of this mutant in the perinuclear endoplasmic reticulum. However, when solubilized with a zwitterionic detergent, this mutant bound [125I]VIP specifically, indicating that it retained intrinsic binding activity. The construction of other mutants in which three out of four N-glycosylation sites were altered, demonstrated that N-glycosylation at either Asn58 or Asn69 is necessary and sufficient to ensure correct delivery of the receptor to the plasma membrane. Further pharmacological studies involving incubation of Cos-7 cells with castanospermine or deoxymannojirimycin immediately after transfection of mutated cDNAs encoding receptors with a single glycosylation site at Asn58 or Asn69 suggested that carbohydrate at Asn58 was involved in a calnexin-dependent folding process of the receptor whereas carbohydrate at Asn69 was not. These studies highlight the functional importance of the N-glycosylation of the human VIP 1 receptor which belongs to a new subfamily of seven membrane-spanning receptors.
通过对人血管活性肠肽（VIP）1受体上Asn58、Asn69、Asn100（N端胞外结构域）和Asn293（第二个胞外环）的四个共有N-糖基化位点进行定点诱变（Asn→Thr），研究了N-连接碳水化合物在该受体中的功能作用。在Cos-7细胞中瞬时表达后，通过配体结合试验、亲和交联、蛋白质印迹以及对表位标记受体蛋白进行共聚焦激光显微镜检查，对突变受体进行了研究。每个共有位点的突变表明，Asn58、Asn69和Asn100被一个9 kDa的N-连接碳水化合物占据而Asn293未用于糖基化。每个突变受体均在Cos-7细胞中表达（蛋白质印迹）并转运至质膜（共聚焦显微镜检查）。它们显示出与野生型受体相似的解离常数，即0.5 - 1 nM。相比之下，由于该突变体被严格隔离在核周内质网中，未观察到缺乏共有N-糖基化位点的突变体与Cos-7细胞有VIP结合。然而，当用两性离子去污剂溶解时，该突变体特异性结合[125I]VIP，表明它保留了内在结合活性。构建四个N-糖基化位点中有三个发生改变的其他突变体，证明Asn58或Asn69处的N-糖基化对于确保受体正确转运至质膜是必要且充分的。进一步的药理学研究涉及在转染编码在Asn58或Asn69处有单个糖基化位点的受体的突变cDNA后，立即用栗精胺或脱氧甘露基野尻霉素孵育Cos-7细胞，结果表明Asn58处的碳水化合物参与了受体的钙连蛋白依赖性折叠过程，而Asn69处的碳水化合物则未参与。这些研究突出了人VIP 1受体N-糖基化的功能重要性，该受体属于七跨膜受体的一个新亚家族。
