An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor.
The effect of N-glycosylation on the assembly of N-methyl-D-aspartate (NMDA) heteromeric cloned receptors was studied. Thus human embryonic kidney (HEK) 293 cells were cotransfected with N-methyl-D-aspartate R1 (NR1) and N-methyl-D-aspartate R2A (NR2A) clones and the cells grown post-transfection in the presence of tunicamycin (TM). TM treatment resulted in a decrease of the NR1 subunit with M(r) 117 000 with a concomitant increase in a M(r) 97 000 immunoreactive species previously identified as the non-N-glycosylated NR1 subunit. In parallel, TM caused a dose-dependent inhibition of [3H]MK801 binding to the expressed receptor which was a result of an approximate four-fold reduction in the Dissociation Constant (KD) but with no change in the number of binding sites (Bmax). NMDA receptor cell surface expression was unchanged following TM treatment but it did result in a decrease in the percentage cell death post-transfection compared to control samples. The removal of TM from the cell culture media resulted in a return to the control KD value for [3H]MK801 binding and partial reglycosylation of newly synthesized NR1 subunit. These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A receptors. Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A receptor.
Chazot
P L
PL
Department of Pharmaceutical Chemistry, School of Pharmacy, London, UK.
Cik
M
M
Stephenson
F A
FA
D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D016291:Dizocilpine Maleate / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism; D006801:Humans; D007668:Kidney; D051379:Mice; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D018076:DNA, Complementary; D018691:Excitatory Amino Acid Antagonists; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; D016291:Dizocilpine Maleate