Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

被引:15
作者
Pasternack, A
Coleman, SK
Féthière, J
Madden, DR
LeCaer, JP
Rossier, J
Pasternack, M
Keinänen, K
机构
[1] Univ Helsinki, Viikki Bioctr, Dept Biosci, Div Biochem, FIN-00014 Helsinki, Finland
[2] Univ Helsinki, Inst Biotechnol, FIN-00014 Helsinki, Finland
[3] Max Planck Inst Med Res, Ion Channel Struct Res Grp, Heidelberg, Germany
[4] Ecole Super Phys & Chim Ind Ville Paris, Lab Neurobiol & Divers Cellulaire, CNRS, Paris, France
关键词
AMPA receptor; glycosylation; ligand-binding domain;
D O I
10.1046/j.1471-4159.2003.01611.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The ligand-binding domains of AMPA receptor subunits carry two conserved N -glycosylation sites. In order to gain insight into the functional role of the corresponding N -glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N -glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.
引用
收藏
页码:1184 / 1192
页数:9
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