N-glycans of sphingosine 1-phosphate receptor Edg-1 regulate ligand-induced receptor internalization.
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.
Kohno
Takayuki
T
Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan.
Wada
Atsushi
A
Igarashi
Yasuyuki
Y
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D004705:Endocytosis; D019286:Endopeptidase K / Q000737:chemistry; D006031:Glycosylation; D017874:Immediate-Early Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008246:Lysophospholipids; D021962:Membrane Microdomains / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D022161:Transport Vesicles / Q000378:metabolism
D017874:Immediate-Early Proteins; D008024:Ligands; D008246:Lysophospholipids; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; C060506:sphingosine 1-phosphate; D001216:Asparagine; D020928:Mitogen-Activated Protein Kinases; D019286:Endopeptidase K; D013110:Sphingosine