Mutagenesis of N-glycosylation sites in the human vasoactive intestinal peptide 1 receptor. Evidence that asparagine 58 or 69 is crucial for correct delivery of the receptor to plasma membrane
Mutagenesis of N-glycosylation sites in the human vasoactive intestinal peptide 1 receptor. Evidence that asparagine 58 or 69 is crucial for correct delivery of the receptor to plasma membrane
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DOI：
10.1021/bi952022h
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发表时间：
1996-02-13
期刊：
影响因子：
2.9
通讯作者：
Laburthe, M
中科院分区：
文献类型：
--
作者：
Couvineau, A;Fabre, C;Laburthe, M
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 Asn(58), Asn(69), Asn(100) (N-terminal extracellular domain) and Asn(293) (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 Asn(58), Asn(69), and Asn(100) were occupied by a 9-kDa N-linked carbohydrate whereas Asn(293) 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 [I-125]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 Asn(58) or Asn(69) 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 Asn(58) or Asn(69) suggested that carbohydrate at Asn(58) was involved in a calnexin-dependent folding process of the receptor whereas carbohydrate at Asn(69) 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.
