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.
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.
Couvineau
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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. couvineau@citi2.fr
Fabre
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Gaudin
P
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Maoret
J J
JJ
Laburthe
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D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D018076:DNA, Complementary; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D001216:Asparagine