title: The integrity of the glycine co-agonist binding site of N-methyl-D-aspartate receptors is a functional quality control checkpoint for cell surface delivery.
abstract: 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 approximately 3 x 10(4) 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.
authors.affiliation: School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.
authors.forename: Anna V
authors.initials: AV
authors.lastname: Kenny
authors.affiliation: School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Cousins
authors.affiliation: School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.
authors.forename: Leonor
authors.initials: L
authors.lastname: Pinho
authors.affiliation: School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom. Electronic address: anne.stephenson@pharmacy.ac.uk.
authors.forename: F Anne
authors.initials: FA
authors.lastname: Stephenson
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D005998:Glycine / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*
chemical_list: D018691:Excitatory Amino Acid Antagonists; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D007736:Kynurenic Acid; C066192:5,7-dichlorokynurenic acid; D005998:Glycine