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