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.
Highlights
Type NR1/NR2A transfectants normalized to 100% cell NR1-1a (D732A)/NR2A NMDA Receptors Are Retained in death, for the NR1-1a mutations, there was no significant the ER; Demonstration by Confocal Microscopy Imaging and deglycosylation using either endoglycosidase H or N-glycosidase F
For NR1-1a (D732A)/NR2A receptors, a strong co- NR2A subunits were co-expressed in HEK 293 cells, immunodistribution for both NR1 and NR2A immunoreactivities with precipitations were carried out using anti-NR1 C2 antibodies immunostaining for the ER marker, calreticulin, was observed, and immune pellets (Fig. 4A, e– h and m–p)
Ary ER quality control points, such as the ER retention sequence found in certain NR1 splice forms, ensure that only assembled NMDA receptors exit the ER, since co-association with NR2 subunits and scaffolding molecules mask the ER retention motif such that forward trafficking can occur
Summary
Functional studies of wild-type and point-mutated subunits expressed in Xenopus oocytes [3,4,5,6,7,8,9,10,11] or mammalian cells [12] have identified within these domains key amino acids that are important mediators for the high affinity binding of glycine [2,3,4,5,6] and glutamate [7,8,9,10,11,12]. For non-NMDA, ionotropic glutamate receptors, additional, functional quality control checkpoints have been shown to contribute to the regulation of cell surface trafficking.
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