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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Asparagine / chemistry
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Cell Membrane / metabolism
  • DNA, Complementary / genetics
  • Glycosylation
  • Humans
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Receptors, Vasoactive Intestinal Peptide / chemistry*
  • Receptors, Vasoactive Intestinal Peptide / genetics*
  • Receptors, Vasoactive Intestinal Peptide / metabolism
  • Transfection
  • Vasoactive Intestinal Peptide / metabolism


  • DNA, Complementary
  • Receptors, Vasoactive Intestinal Peptide
  • Vasoactive Intestinal Peptide
  • Asparagine