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

Arja Pasternack, Sarah K. Coleman, James Féthière, Dean R. Madden, Jean-Pierre LeCaer, Jean Rossier, Michael Pasternack and Kari Keinänen 2003. Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain. Journal of Neurochemistry. 84 (5), pp. 1184-1192. https://doi.org/10.1046/j.1471-4159.2003.01611.x

TitleCharacterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain
TypeJournal article
AuthorsArja Pasternack, Sarah K. Coleman, James Féthière, Dean R. Madden, Jean-Pierre LeCaer, Jean Rossier, Michael Pasternack and Kari Keinänen
Abstract

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.

JournalJournal of Neurochemistry
Journal citation84 (5), pp. 1184-1192
ISSN0022-3042
Year2003
PublisherWiley
Digital Object Identifier (DOI)https://doi.org/10.1046/j.1471-4159.2003.01611.x
Publication dates
PublishedMar 2003

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