Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain.
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.
Pasternack
Arja
A
Viikki Biocenter, Department of Biosciences (Division of Biochemistry), Viikinkaari 5D, FIN-00014 University of Helsinki, Finland.
Coleman
Sarah K
SK
Féthière
James
J
Madden
Dean R
DR
LeCaer
Jean-Pierre
JP
Rossier
Jean
J
Pasternack
Michael
M
Keinänen
Kari
K
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D002460:Cell Line; D002942:Circular Dichroism; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017470:Receptors, Glutamate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D014415:Tunicamycin / Q000494:pharmacology
D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D011134:Polysaccharides; D021122:Protein Subunits; D017470:Receptors, Glutamate; D011994:Recombinant Proteins; C109433:glutamate receptor type D; D014415:Tunicamycin; C085706:FLAG peptide