The distal carboxyl terminal of rat NR3B subunit regulates NR1-1a/NR3B and
NR1-2a/NR3B surface trafﬁcking
Karen Siaw-Ling Wee
, Zhen-Ning Wee
, Nicholas Boon-How Chow
, Chian-Ming Low
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore
Department of Anaesthesia, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore
Neurobiology and Ageing Programme, Life Sciences Institute, National University of Singapore, Republic of Singapore
-aspartate (NMDA) receptors are complexes assem-
bled from NR1 subunits, NR2 subunits (NR2A-D) and/or NR3
subunits (NR3A-B) (Dingledine et al., 1999). Each subunit
comprises an extracellular N-terminus, four membrane domains
(M1–M4), and an intracellular C-terminus.
Endoplasmic reticulum (ER) retention signals mediate reten-
tion of improperly folded or assembled polypeptides. They are
present in the C1 cassette of NR1 and M3 region of NR1 and NR2B
subunits (Horak et al., 2008; Horak and Wenthold, 2009). Masking
of these signals is required for the release of subunits from the ER
(Standley et al., 2000; Hawkins et al., 2004). During co-assembly,
inter-subunit interactions bring subunits into close proximity;
thereby allowing the masking of ER retention signals (Zerangue
et al., 1999; Standley et al., 2000; Hawkins et al., 2004). Although
the amino-terminal domain (ATD) is sufﬁcient for assembly and
oligomerization (Meddows et al., 2001; Horak et al., 2008), more
distal regions of the subunits may subsequently interact to result
in physical masking of retention motifs in M3 and C-terminus of
NR1 and NR2B (Schorge and Colquhoun, 2003; Horak et al., 2008).
Most studies to-date have focused on the trafﬁcking signals
presentin NR1 and NR2B subunits (Okabe et al.,1999; Hawkins et al.,
2004; Horak et al., 2008). There is a lack of knowledge pertaining to
trafﬁcking signals in NR3. The precise motif(s) responsible for the
trafﬁcking of NR3B-containing receptors from ER to the cell surface
remain unclear although a previous study had identiﬁed the region
between amino acid residues 952 and 985 in the C-terminus of
mouse NR3B as a critical determinant for cell surface expression of
NR1-1a/NR3B receptors (Matsuda et al., 2003).
In this study, we focused on the distal C-terminus of rat NR3B
(rNR3B) and investigated the role of a conserved RERLR motif
within this region. We also investigated a plausible role of NR3B C-
terminus in masking ER retention signal(s) on NR1 subunit by co-
expressing the full length and truncated rNR3B separately with
rNR1 splice variants that possess or lack the C1 cassette.
2. Materials and methods
2.1. Molecular cloning
The rat NR3B clone (rNR3B) was kindly provided by Dr. Zhang, D. Burnham
Institute, La Jolla, CA, USA. The rNR3B-HA construct was generated by adding a
hemagglutinin (HA) tag to the distal end of the C-terminus as described elsewhere
previously (Matsuda et al., 2003). rNR3B C-terminal truncation constructs were
Neurochemistry International 57 (2010) 97–101
Received 24 March 2010
Received in revised form 3 May 2010
Accepted 4 May 2010
Available online 11 May 2010
-aspartate (NMDA) receptors are multi-subunit receptors formed from assembly of NR1 with
NR2 and/or NR3 subunits. In this study, we investigated the role of a conserved RERLR motif present in a
region within the distal carboxyl terminal of rat NR3B (between residues 952 and 984) in targeting NR1-
1a/NR3B and NR1-2a/NR3B receptors to the cell surface. Surface biotinylation, confocal immunoﬂuo-
rescence microscopy and site-directed mutagenesis studies showed RERLR motif does not inﬂuence the
surface expression of NR1-1a/NR3B NMDA receptor complex. Our bioinformatics analysis further
showed this region can also exist as a coiled-coil domain. Truncation of this putative coiled-coil domain
in NR3B affects surface expression of NR1-1a/NR3B and NR1-2a/NR3B receptors similarly suggesting
that NR1 C1 cassette is not involved in the effect mediated by the distal carboxyl region of NR3B. This
study represents the ﬁrst attempt to evaluate a speciﬁc motif in regulating rat NR3B surface expression.
ß 2010 Elsevier Ltd. All rights reserved.
Abbreviations: HEK, human embryonic kidney cells; ER, endoplasmic reticulum;
ATD, amino-terminal domain; M4, transmembrane domain 4; HA, hemagglutinin;
PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacryl-
amide gel electrophoresis; EDTA, ethylene diamine tetraacetic acid; BSA, bovine
* Corresponding author at: Centre for Life Sciences (CeLS), #04-06, 28 Medical
Drive, National University of Singapore, S117456, Republic of Singapore.
Tel.: +65 6516 5841; fax: +65 6873 7690.
E-mail address: firstname.lastname@example.org (C.-M. Low).
Equal contribution from authors.
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0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved.