N-glycans of sphingosine 1-phosphate receptor Edg-1 regulate ligand-induced receptor internalization

FASEB J. 2002 Jul;16(9):983-92. doi: 10.1096/fj.01-0809com.

Abstract

Endothelial differentiation gene-1 product (Edg-1) is a G-protein-coupled receptor (GPCR) for the platelet derived bioactive lipid mediator sphingosine 1-phosphate (Sph-1-P). Recent studies have shown that in response to Sph-1-P, Edg-1 mediates various signaling pathways through downstream signaling molecules, such as MAP kinase and calcium, via heterotrimeric G-proteins. We found for the first time that Edg-1 is glycosylated in its amino-terminal extracellular portion, and further identified the specific glycosylation site as asparagine 30 by creating a nonglycosylated mutant of Edg-1 (N30D-Edg-1) and transfecting it into cell lines. The nonglycosylated mutant receptors, resembling their wild-type controls, were predominantly expressed in the plasma membrane. Although there was no difference in ligand binding ability and ligand-induced MAP kinase activation in the wild-type and mutant receptors, nonglycosylated Edg-1 was much less responsive for ligand-induced internalization. Unlike the wild-type receptor, which was associated with the caveolae, nonglycosylated N30D-Edg-1 was dispersed broadly in the membrane fractions separated by sucrose density gradient centrifugation, suggesting that internalization and microdomain localization of N-glycosylated Edg-1 might be related. Although the precise molecular mechanism of the internalization of the N-glycosylated Edg-1 localized in the microdomain remains to be examined, the present study suggested that the presence of N-linked glycan in the receptor may play a regulatory role in the receptor dynamics in ligand-stimulated mammalian cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Asparagine / metabolism
  • CHO Cells
  • Cell Membrane / chemistry
  • Cricetinae
  • Endocytosis*
  • Endopeptidase K / chemistry
  • Glycosylation
  • Immediate-Early Proteins / analysis
  • Immediate-Early Proteins / chemistry*
  • Immediate-Early Proteins / metabolism*
  • Kinetics
  • Ligands
  • Lysophospholipids*
  • Membrane Microdomains / chemistry
  • Mice
  • Microscopy, Fluorescence
  • Mitogen-Activated Protein Kinases / metabolism
  • Molecular Sequence Data
  • Polysaccharides / metabolism*
  • Protein Transport
  • Receptors, Cell Surface / analysis
  • Receptors, Cell Surface / chemistry*
  • Receptors, Cell Surface / metabolism*
  • Receptors, G-Protein-Coupled*
  • Receptors, Lysophospholipid
  • Sphingosine / analogs & derivatives*
  • Sphingosine / pharmacology
  • Transport Vesicles / metabolism

Substances

  • Immediate-Early Proteins
  • Ligands
  • Lysophospholipids
  • Polysaccharides
  • Receptors, Cell Surface
  • Receptors, G-Protein-Coupled
  • Receptors, Lysophospholipid
  • sphingosine 1-phosphate
  • Asparagine
  • Mitogen-Activated Protein Kinases
  • Endopeptidase K
  • Sphingosine