An Endoplasmic Reticulum Retention Signal Located in the Extracellular Amino-terminal Domain of the NR2A Subunit of N-Methyl-d-aspartate Receptors

Abstract

N-Methyl-D-aspartate (NMDA) receptors play critical roles in complex brain functions as well as pathogenesis of neurodegenerative diseases. There are many NMDA isoforms and subunit types that, together with subtype-specific assembly, give rise to significant functional heterogeneity of NMDA receptors. Conventional NMDA receptors are obligatory heterotetramers composed of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. When individually expressed in heterogeneous cells, most of the NR1 splice variants and the NR2 subunits remain in the endoplasmic reticulum (ER) and do not form homomeric channels. The mechanisms underlying NMDA receptor trafficking and functional expression remain uncertain. Using truncated and chimeric NMDA receptor subunits expressed in heterogeneous cells and hippocampal neurons, together with immunostaining, biochemical, and functional analyses, we found that the NR2A amino-terminal domain (ATD) contains an ER retention signal, which can be specifically masked by the NR1a ATD. Interestingly, no such signal was found in the ATD of the NR2B subunit. We further identified the A2 segment of the NR2A ATD to be the primary determinant of ER retention. These findings indicate that NR2A-containing NMDA receptors may undergo a different ER quality control process from NR2B-containing NMDA receptors. Ionotropic glutamate receptors (iGluRs)2 mediate most of the excitatory neurotransmission in the central nervous system. They play key roles in complex brain functions as well as in the pathogenesis of neurodegenerative diseases. Based on pharmacological properties and sequence similarities, iGluRs can be grouped into three major subtypes: GluR1 to -4 subunits form ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, GluR5 to -7 and KA1 and -2 subunits make up kainate receptors, and NR1 together with NR2A to -D subunits comprise the NMDA receptors (1). All iGluR subunits share a unique membrane topology with a large extracellular NH 2 -terminal domain, three transmembrane segments (TM1 (transmembrane domain 1), TM3, and TM4), a P-loop region, and a cytoplasmic COOH terminus (2, 3). Based on the sequence homology to bacterial periplasmic binding proteins, the NH 2 -terminal domain of iGluRs can be divided into two domains in tandem: the amino-terminal domain (ATD), which includes the first 400 or so amino acids (4), and the following S1 domain preceding TM1, which forms the ligand-binding domain together with the extracellular loop between TM3 and TM4 (S2 domain) (5, 6).Among iGluRs, NMDA receptors are special in that conventional NMDA receptors are obligatory tetrameric membrane proteins composed of two glycine-binding NR1 and two glutamate-binding NR2 subunits. The NR1 subunit is essential for the formation of functional NMDA receptor channel, whereas the NR2 subunit modifies channel properties, such as current kinetics and channel conductance (1). The major NR1 splice variant and the NR2 subunits are retained in the ER when expressed alone in heterogeneous cells. On...

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…Data suggest that the ATD plays a crucial role in receptor oligomerization and perhaps trafficking (Kuusinen et al, 1999; Leuschner and Hoch, 1999; Ayalon and Stern-Bach, 2001; Ayalon et al, 2005;Qiu et al, 2009). The interaction between the ATDs is sufficient to allow isolated ATDs to form stable dimers in solution (Clayton et al, 2009;Jin et al, 2009;Kumar et al, 2009).…”

Adaptor Protein APPL1 Couples Synaptic NMDA Receptor with Neuronal Prosurvival Phosphatidylinositol 3-Kinase/Akt Pathway

“…The fluorescence imaging work station for fluorescence resonance energy transfer (FRET) and the FRET quantification method have been described previously (Qiu et al, 2005(Qiu et al, , 2009. Briefly, the fluorescence imaging workstation consisted of an inverted microscope (TE2000; Nikon), Dual-View (Optical Insights), and a SNAP-HQ-cooled CCD (Roper Scientific).…”

“…4A). FRET is another method to detect protein-protein interactions in vivo (Qiu et al, 2005(Qiu et al, , 2009). Here, we tagged APPL1 with CFP and PSD95 with YFP and examined the interaction of APPL1 with PSD95 using FRET (Fig.…”

Transmembrane Region of N-Methyl-d-aspartate Receptor (NMDAR) Subunit Is Required for Receptor Subunit Assembly

“…Studies using biochemical methods and immunostaining indicate that the C terminus and/or M4 are not involved in the heteromer formation between GluN1 and GluN2 subunits (9,15), whereas the ATD or even the whole N terminus is necessary for NMDAR subunit assembly (15,16). However, still other research indicates that the ATD mainly plays modulatory roles in NMDAR trafficking and function and is not directly involved in receptor assembly (10,17,18).…”