Kenny, A.V., Cousins, S.L., Pinho, L. and Stephenson, F.A. (2009) The integrity of the glycine co-agonist binding site of NMDA receptors is a functional quality control check-point for cell surface delivery. Journal of Biological Chemistry, 284 . pp. 324-333. 10.1074/jbc.M804023200.
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N-Methyl-d-aspartate receptors are a subclass of ligand-gated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-d-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an ∼3 × 104 decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors.
|Additional Information:||Full text available electronically from the School of Pharmacy Library.|
|Departments, units and centres:||Department of Pharmaceutical and Biological Chemistry > Department of Pharmaceutical and Biological Chemistry|
|Journal or Publication Title:||Journal of Biological Chemistry|
|Deposited By:||Library Staff|
|Deposited On:||27 Jan 2010 13:43|
|Last Modified:||24 Nov 2011 14:32|
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