Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

Abstract

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors. KeywordsER retention; glutamate receptor; ion channel; masking; patch-clamp; recombinant; subunit assembly N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ionotropic receptors mediating excitatory neurotransmission in the central nervous system.1 It is believed that these receptors are heterotetramers composed of two NR1 and two NR2 and/or NR3 subunits.2 All NMDA receptor subtypes share the same structural topology with three membrane domains (M1, M3 and M4), a re-entrant pore-forming domain (M2), and extracellular (N-terminus and loop between M3 and M4) and intracellular (C-terminus) regions. The most abundant NR1 splice variant (NR1-1) and all NR2 subunits are retained in the ER when expressed by themselves in various heterologous cells and neurons; however, co-expression of NR1-1 and NR2 leads to surface expression of assembled complexes.3 -5 Thus, these data suggest the presence of a mechanism(s) regulating ER retention of unassembled subunits, correct assembly of the subunits into functional receptors, as well as release of the functional receptors from the ER.There have been a number of reports studying the role of ER retention in the assembly of NMDA receptors. First, the localization of ER retention signals was studied using chimeric proteins composed of tac (an interleukin-2 receptor subunit) and NMDA receptor C-termini. 6-9 Since both NR1-1 and NR2B C-termini cause the chimeric protein to be retained in the ER, an extensive mutagenesis study was performed. Hence, an RXR ER retention motif in the NR1-1 C-terminus was identified. Efforts to localize a particular motif responsible for the ER retention of tac-NR2B chimera have been unsuccessful. Second, using truncated and chimeric NMDA receptor subunits, we showe...

ER to synapse trafficking of NMDA receptors

“…However, a later study proposed that the HLFY motif is not necessary for the formation of the surface functional NMDAR, as it can be replaced by alanines if the C-terminus is absent (Yang et al, 2007 ). Similarly, the deletion of the GluN2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the GluN2B subunit, GluN2B truncated before M4 domain and GluN2B M4 domain, were co-expressed together with the GluN1 subunit (Horak et al, 2008a ). Together, these data indicate that the HLFY motif may provide a structural role to ensure the proper orientation of the membrane domains and/or the C-termini in the ER processing of the GluN1/GluN2 receptors.…”

Independent regulation of early trafficking of NMDA receptors by ligand-binding domains of the GluN1 and GluN2A subunits

“…Previous studies identified other regions in GluN subunits besides LBDs, including ATD (Qiu et al, 2009), M3 domains (Horak et al, 2008), and CTDs (Standley et al, 2000; Scott et al, 2001; Xia et al, 2001; Horak and Wenthold, 2009), containing ER retention and export signals. Therefore, we anticipate that future studies will investigate at what level other ER retention and export signals contribute to regulating the surface expression of the GluN1/GluN2 receptors with mutated LBDs.…”

Protein quality control of N-methyl-D-aspartate receptors

“…For example, the HLFY motif on the end of TM4 in the GluN2B subunit, which is not present in GluN1 subunits, was found to likely be involved in the export of NMDARs (Hawkins et al, 2004). Replacing each of these key residues with alanine, or truncating the GluN2B before the TM4 domain was found to disturb the surface trafficking of functional receptors (Yang et al, 2007;Horak et al, 2008a). It is believed that this motif may play a role as a conformational signal, ensuring proper orientation of the receptor domains.…”

“…The key amino acids Trp 636 and Tyr 647/Thr 648 within the GluN1 TM3 and Trp 635 and Ser 645/Tyr 646/Thr 647 within GluN2B TM3 have been shown to regulate their surface delivery. These residues ensure subunits are retained in the ER until correct conformation of the TM3 domains is achieved to continue the forward trafficking (Horak et al, 2008a;Kaniakova et al, 2012).…”