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TARP γ-8 glycosylation regulates the surface expression of AMPA receptors

Article  in  Biochemical Journal · December 2014

DOI: 10.1042/BJ20140806 · Source: PubMed

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Biochem. J. (2015) 465, 471–477 (Printed in Great Britain) doi:10.1042/BJ20140806
TARP γ -8 glycosylation regulates the surface expression of AMPA receptors
Chan-Ying Zheng*, Kai Chang*, Young Ho Suh†1 and Katherine W. Roche*1
*Receptor Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, U.S.A.
†Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799, South Korea
TARP [transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid) receptor regulatory protein] γ -8 is an
auxiliary subunit of AMPA receptors that is widely distributed
in the hippocampus. It has been shown that TARP γ -8 promotes
surface expression of AMPA receptors; however, how TARP γ -8
regulates the expression of AMPA receptors remains unclear. In
the present study, we examined the effect of TARP glycosylation
on AMPA receptor trafficking. We first showed that TARP γ -8
is an N-glycosylated protein, which contains two glycosylation
sites, Asn53 and Asn56 , and compared this with the glycosylation
of TARP γ -2 and the AMPA receptor auxiliary protein CNIH-2
(cornichon homologue 2). We next examine the effect of TARP
glycosylation on TARP trafficking and also on AMPA receptor
surface expression. We find that TARP γ -8 glycosylation is
INTRODUCTION
AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid)-type ionotropic glutamate receptors primarily mediate fast
excitatory synaptic transmission and long-lasting plasticity in
the central nervous system [1]. AMPA receptors are tetramers
composed of combinations of GluA1–GluA4 subunits assembled
either as GluA1/GluA2 or GluA2/GluA3 heteromers in the
hippocampus [2,3]. In addition, TARPs (transmembrane AMPA
receptor regulatory proteins) are required as essential auxiliary
subunits, which regulate maturation, surface expression and
channel gating of AMPA receptors [4–9].
TARPs are tetraspan transmembrane proteins that modulate
the biophysical properties and subcellular distribution of AMPA
receptors. TARP binding to the postsynaptic scaffolding protein
PSD-95 via the TARP extreme C-terminal domain is critical
for its effect on synaptic AMPA receptors [10,11]. TARP γ -2
(Cacng2), known as stargazin, is the canonical TARP that is highly
expressed in cerebellum. It was identified from the spontaneous
mutant mouse stargazer in which functional glutamate receptors
are lacking in cerebellar granule neurons [11–13]. In contrast,
TARP γ -8 is preferentially expressed in the hippocampus at both
synaptic and extrasynaptic sites [14–16]. Recently, a new family
of AMPA receptor auxiliary subunits, the cornichon homologues
(CNIH-2/3), have been characterized, which regulate trafficking,
gating and pharmacology of AMPA receptors [17–19].
Assembly and stoichiometry of multisubunit protein complexes
are regulated by a stringent quality control system in the
ER (endoplasmic reticulum). In particular, the hydrophilic N-
glycans can often mediate protein quality control by regulating
proper protein folding, the removal of misfolded proteins,
intracellular trafficking, and stabilization of protein conformation
Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CNIH, cornichon homologue; DIV, days in vitro ; Endo Hf, endoglycosidase
Hf; ER, endoplasmic reticulum; HEK, human embryonic kidney; IRES, internal ribosome entry site; NGS, normal goat serum; PNGase F, peptide N-
glycosidase F; TARP, transmembrane AMPA receptor regulatory protein; Ub, ubiquitin; WT, wild-type.
1
Correspondence may be addressed to either of these authors (email suhyho@snu.ac.kr or rochek@ninds.nih.gov).
471
www.biochemj.org
critical for surface expression of both TARP γ -8 and GluA1 in
heterologous cells and neurons. Specifically, knockdown of TARP
γ -8 causes a decrease in both total and surface AMPA receptors.
We find that the expression of unglycosylated TARP γ -8 in
cultured neurons is unable to restore GluA1 expression fully.
Furthermore, when the maturation of TARP γ -8 is impaired, a
large pool of immature GluA1 is retained intracellularly. Taken
together, our data reveal an important role for the maturation of
TARP γ -8 in the trafficking and function of the AMPA receptor
complex.
Key words: AMPA receptor, CNIH-2, N-linked glycosylation,
TARP γ -2, TARP γ -8, trafficking.
and subunit assembly [20]. N-glycans are recognized by
the ER lectin-like chaperones calnexin, calreticulin and BiP
(immunoglobulin heavy-chain-binding protein), which facilitate
Biochemical Journal
glycoprotein folding and assembly. Correctly assembled proteins
are released and transported to the Golgi apparatus. If N-
glycosylation is prevented, proteins that are incompletely folded
or assembled are generally retained in the ER, leading to
aggregation and/or proteasomal degradation [21]. TARP γ -
2 facilitates the export of AMPA receptors from the ER.
The N-glycosylation of AMPA receptors has been shown to
be incomplete in the cerebellum of stargazer mice, reflecting
immature AMPA receptors [22]. In addition, total protein levels
of AMPA receptors are markedly reduced in the hippocampus
of TARP γ -8-knockout mice without a change in mRNA level,
suggesting that TARP γ -8 is likely to be required for proper
assembly and quality control of AMPA receptor complexes in the
ER [8,14,19,23]. However, there is no direct evidence regarding a
potential role for the N-glycosylation of TARPs in regulating the
functional expression of AMPA receptors.
In the present study, we have characterized the N-glycosylation
of TARP γ -8 and compared this with that of TARP γ -2 and
CNIH-2. We found that the unglycosylated TARP γ -8 mutant
is retained in a distinct intracellular compartment and is less
efficiently expressed on the plasma membrane in heterologous
cells. In addition, surface expression of GluA1 is impaired
when co-expressed with the unglycosylated TARP γ -8 mutant.
In particular, expression of unglycosylated TARP γ -8 on the
knockdown background is unable to restore total and surface
expression of GluA1 that is markedly reduced in TARP γ -8-
knockdown neurons. Furthermore, N-glycosylation of GluA1 is
reduced by the expression of unglycosylated TARP γ -8. In sharp
contrast, CNIH is not glycosylated at all. Taken together, our

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472 C.-Y. Zheng and others
data show that proper maturation of TARP γ -8 is necessary for
trafficking and function of the AMPA receptor complex.
MATERIALS AND METHODS
Glycosidase assay and Western blotting
Primary rat hippocampal neurons or HEK (human embryonic
kidney)-293T cells were washed three times with ice-cold PBS
and harvested in TNE buffer (50 mM Tris/HCl, pH 8.0, 150 mM
NaCl, 2 mM EDTA, and protease and phosphatase inhibitor
cocktails). The cells were sonicated for 10 s to disrupt the plasma
membranes and centrifuged at 200 000 g for 30 min. The resulting
pellet was resuspended in TNE buffer, and solubilized with a final
concentration of 1 % (w/v) SDS and 1 % (v/v) Triton X-100.
The lysates were incubated on ice for 30 min and centrifuged at
20 000 g for 15 min at 4 ◦ C to remove insoluble materials. For
the glycosidase assays, the lysates were incubated with Endo
Hf (endoglycosidase Hf) (1500 units) or PNGase F (peptide N-
glycosidase F) (750 units) overnight at 37 ◦ C. The glycosidase-
treated samples were mixed with 6× Laemmli buffer, incubated
for 20 min at 37 ◦ C, resolved by SDS/PAGE and analysed by
Western blotting. Densitometry was used to quantify the ratio
of immature to total (mature plus immature) GluA1 subunits
using ImageJ software (NIH). Anti-GluA1 (MAB2263), anti-
TARP γ -2 (AB9876) and anti-TARP γ -8 (07-577) antibodies
were obtained from Millipore. Anti-α-tubulin antibody was from
Sigma. Horseradish-peroxidase-conjugated secondary antibodies
were incubated and detected using ECL substrate (Thermo
Scientific).
Immunostaining
COS-7 (fibroblast-like monkey kidney) cells were transfected
on the day of splitting using LipofectamineTM 2000 (Invitrogen)
[24]. After 2 days, cells were fixed with a mixture of 4 % (w/v)
paraformaldehyde and 4 % (w/v) sucrose at room temperature
for 10 min. After washing with PBS, cells were permeabilized
in 0.25 % Triton X-100 for 5 min. The cells on coverslips
were moved into a PELCO BioWave® Pro Microwave system.
Microwave power was set to 150 W. Cells were blocked in PBS
containing 10 % (v/v) NGS (normal goat serum) for a total of
5 min (2 min on, 1 min off, 2 min on) in the microwave oven.
The cells were then incubated with primary antibody in PBS
containing 3 % (v/v) NGS for 8 min (3 min on, 2 min off, 3 min
on). The primary antibody was removed, and the cells were
rinsed three times in PBS. The cells were then incubated with
FITC-, Cy3- (indocarbocyanine) or Cy5- (indodicarbocyanine)
conjugated secondary antibody for a total of 8 min (3 min on,
2 min off, 3 min on). After washing three times with PBS, cells
were mounted on slides using ProLong gold antifade reagent
(Invitrogen) and imaged using a Zeiss 510 confocal microscope.
When labelled for surface and total GluA1, 21 DIV (days
in vitro) hippocampal neurons were incubated with anti-(GluA1
N-terminus) antibody (Millipore) for 30 min at 37 ◦ C, and fixed
at room temperature for 10 min, then immunostained using a
microwave oven.
Confocal imaging
All images were captured using a Zeiss LSM 510 confocal
microscope with a ×63 objective, using ×1.5 optical zoom and a
1024 pixel×1024 pixel resolution. The mean intensity of surface
staining was measured as described in [25]. For each neuron, three
independent secondary dendritic branches with a total length of at
least 30 μm were used for determining a mean intensity of surface

c The Authors Journal compilation 
c 2015 Biochemical Society
and total GluA1 staining. In total, 5–15 pyramidal neurons from
three independent transfections were quantified using MetaMorph
software. Line fluorescence intensity was measured by Zeiss LSM
510 image examiner software.
Surface biotinylation assay
Primary hippocampal neurons or HEK-293T cells were
washed three times with PBS containing 1 mM MgCl2 and
0.1 mM CaCl2 (PBS + + ), and incubated with membrane-
impermeant EZ-Link sulfo-NHS-SS-biotin [sulfosuccinimidyl-
2-(biotinamido)ethyl-1,3-dithiopropionate] (Thermo Scientific)
in PBS for 20 min at 4 ◦ C with gentle rocking as described
previously [26]. The biotinylated cell membrane fraction was
prepared as described above. The supernatants were incubated
with NeutrAvidin–agarose resin (Thermo Scientific) for 3 h at
4 ◦ C with gentle rotation. The resins were washed and the bound
proteins were analysed by Western blotting.
Cell culture and lentivirus infection
All animal procedures were performed in accordance with
the guidelines of the National Institutes of Health Animal
Research Advisory Committee under NINDS (National Institute
of Neurological Disorders and Stroke) protocol 1171. Primary
hippocampal neurons were prepared from E (embryonic day) 18
fetal Sprague–Dawley rat hippocampi. Neurons were plated on
poly-D-lysine-(Sigma) coated dishes or coverslips. For lentiviral
infection, FHUGW lentiviral vector was utilized [27]. To
knockdown endogenous TARP γ -8, 19 nucleotides of murine
TARP γ -8 (target sequence: GCACTGACTACTGGCTCTA) with
a short hairpin and antisense nucleotides was cloned under the
H1 promoter. To simultaneously rescue TARP γ -8 expression,
TARP γ -8 WT (wild-type) or TTAA mutant cDNA containing an
shRNA-resistant sequence (GCACcGAtTAtTGGtTCTA; mutated
residues are indicated in lower-case) was cloned under an
Ub (ubiquitin) promoter. To produce lentivirus particles, HEK-
293T cells were plated in a 100-mm-diameter culture dish and
co-transfected with 8.9, VSV-G (vesicular stomatitis virus
glycoprotein) and lentiviral vectors using X-tremeGENE HD
reagent (Roche). After 36 h of incubation, the culture supernatant
was harvested, centrifuged at 1800 g for 15 min at 4 ◦ C to
remove cell debris and frozen at − 80 ◦ C. For biochemistry
experiments, neurons were infected at 7 DIV using lentiviruses
and then collected for experiments 10 days after infection. For
immunostaining, neurons were infected twice at both 7 and 9
DIV and then used for experiments at 21 DIV.
RESULTS
TARP γ -8 is N-glycosylated at Asn53 and Asn56 , and TARP γ -2 at
Asn48
TARP γ -8 is highly localized on the neuronal surface after
first trafficking through the biosynthetic pathway from the ER–
Golgi to the plasma membrane [22]. To characterize the N-
glycosylation of endogenous TARP γ -8 and γ -2, we utilized
PNGase F, which is an amidase that cleaves all asparagine-linked
(N-linked) oligosaccharides from glycoproteins. This enabled
us to compare the protein mass of TARP with or without
glycosidase treatment to determine whether it contains N-linked
carbohydrates. We found that both endogenous TARP γ -8 and
γ -2 are N-glycosylated, as PNGase F treatment led to a reduction
in protein size (Figure 1A). Because glycosylation maturation
occurs in the ER/cis-Golgi, Endo Hf is a common way to assess
protein maturation to evaluate whether glycosylation is mature.
 TARP γ -8 regulates AMPA receptor trafficking 473

Figure 1 TARP γ -8 Asn53 and Asn56 , and TARP γ -2 Asn48 are N-glycosylated, but CNIH is not
(A) Membrane proteins from primary cultured neurons were treated with Endo Hf or PNGase F, and the N-glycosylation status of the endogenous TARP γ -8 and TARP γ -2 was analysed. (B)
HEK-293T cells were transfected with either vector (Vec-Tf) or CNIH-2 (CNIH-2-Tf) and the lysates were treated with glycosidase. The N-glycosylation status of endogenous CNIH-2 from primary
cultured neurons was also analysed by glycosidase treatment. (C) The consensus glycosylation sites in the first extracellular loops of TARP family members are aligned. The N-glycosylation sites
are highly conserved. (D) A PNGase F assay was performed to identify the N-glycosylation residues. HEK-293T cells were transfected either with TARP γ -8 WT or unglycosylated mutants, and the
lysates were treated with PNGase F overnight at 37 ◦ C. The lysates were resolved by SDS/PAGE, transferred on to a PVDF membrane, and analysed by Western blotting with anti-TARP γ -8 antibody.
(E) PNGase F assay from HEK 293T cells transfected with TARP γ -2 WT or T50A unglycosylated mutant. Molecular masses are indicated in kDa.

When using Endo Hf, which cleaves high-mannose ‘immature’
N-linked oligosaccharides, both endogenous TARP γ -8 and γ -2
were resistant to Endo Hf, consistent with endogenous TARPs
being mature and having exited the ER (Figure 1A). In contrast,
CNIH-2 expressed in HEK-293T cells or endogenous CNIH-2
from cultured neurons was not sensitive to Endo Hf or PNGase F,
showing that CNIH-2 is not glycosylated (Figure 1B).
The NXS/T motif, where X is any amino acid except proline,
is a consensus N-linked protein glycosylation motif. To predict
the potential glycosylation sites in TARPs, we first analysed the
protein sequence of TARP γ -8 and identified two NXS/T motifs,
N53 TT and N56 LT (Figure 1C), which are located within the
first extracellular loop of TARP γ -8. TARP γ -2 also has a
single potential glycosylation motif, N48 ET (Figure 1C) [28].
Interestingly, we found at least one of these potential N-
glycosylation sites was conserved within the first extracellular
loop among other TARP family members (Figure 1C). To test
whether these residues are actually glycosylated, we mutated
TARP γ -8 N53 TT and N56 LT to NTA and NLA respectively (γ -8
T55A and γ -8 T58A). When expressed in HEK-293T cells, we
found that TARP γ -8 WT resulted in diffuse bands on a Western
blot when staining with an anti-TARP antibody. After PNGase F
treatment, a clear band with a lower apparent molecular mass of
∼40 kDa was detected. TARP γ -8 T55A and γ -8 T58A displayed
a similar molecular mass, which is smaller than that of TARP
γ -8 WT, but bigger than that of PNGase F-treated TARP γ -8
(Figure 1D). These results indicate that both Asn53 and Asn56
are glycosylated. Next, we eliminated both glycosylation motifs
by replacing both Thr55 and Thr58 with alanine residues (TARP
γ -8 TTAA). We observed that the size of TARP γ -8 TTAA by
immunoblotting was identical with that of the PNGase F-treated
TARP γ -8, demonstrating that TARP γ -8 has two glycosylation
sites, Asn53 and Asn56 , and that both motifs are utilized for
glycosylation.
In the same way, we mutated the threonine residue within
the consensus motif of TARP γ -2 to alanine to disrupt the
glycosylation of TARP γ -2. HEK-293T cells were transfected
with TARP γ -2 WT or T50A, and incubated with or without
PNGase F. Our results showed that both PNGase F treated
and untreated TARP γ -2 T50A have the same molecular mass
bands. These bands have a smaller apparent molecular mass than
untreated TARP γ -2 WT, and are the same size as PNGase F-
treated TARP γ -2 WT (Figure 1E), indicating that Asn48 is the
sole residue for N-glycosylation of TARP γ -2.
The TARP γ -8 unglycosylated mutant accumulates in intracellular
compartments in heterologous cells
When TARP γ -8 WT was expressed in heterologous cells, we
found that TARP γ -8 is highly expressed on the cell surface
after immunostaining with a specific anti-TARP γ -8 antibody
(Figure 2A). Both TARP γ -8 T55A and γ -8 T58A showed
similar expression patterns to that of TARP γ -8 WT with clear
immunostaining around the edge of the cell. Both WT and single
unglycosylated mutants were localized both in the cytoplasm and
on the cell surface, and sometimes concentrated in the Golgi
apparatus (Figure 2A). Interestingly, unlike TARP γ -8 WT and
single unglycosylated TA mutants, the double unglycosylated
mutant, TARP γ -8 TTAA, was largely clustered intracellularly
in a compartment other than the Golgi apparatus and was largely
absent from the edges of cells (Figure 2A), suggesting the
TARP γ -8 TTAA mutant is likely to be impaired in its forward
trafficking.
It has been shown that TARPs bind directly to both GluA1
and CNIH-2 [29]. To examine whether the binding between
TARP γ -8 and GluA1 or CNIH-2 are preserved in the TARP
γ -8 unglycosylated mutant, HEK-293T cells were co-transfected


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474 C.-Y. Zheng and others
Figure 2 Intracellular localization and binding properties of the TARP γ -8
unglycosylated TTAA mutant in heterologous cells
(A) TARP γ -8 WT or unglycosylated mutants were transfected in COS7 cells and immunostained
with anti-TARP γ -8 antibody. The images were acquired as described in the Materials and
methods section. Representative images are shown. (B) Both TARP γ -8 WT and unglycosylated
γ -8 TTAA mutant bind to CNIH-2 and GluA1. GluA1 and CNIH-2 were co-transfected either
with TARP γ -8 WT or TTAA mutants in HEK-293T cells. The lysates were immunoprecipitated
(IP) with mouse anti-TARP antibody and Western blotting was carried out with the indicated
antibodies. Arrowhead indicates immunoglobulin heavy chain (IgH).
with TARP γ -8, GluA1 and CNIH-2 cDNA, and TARP γ -8 was
immunoprecipitated. We found that TARP γ -8 unglycosylated
TTAA mutant could still bind to GluA1 and CNIH-2 (Figure 2B).
Surface expression of GluA1 is partially impaired by
unglycosylated TARP γ -8 in heterologous cells
Our imaging analysis showed that TARP γ -8 TTAA mutant
is retained in an intracellular compartment; however, any role
TARP γ -8 might have in the surface trafficking of GluA1 was not
known. Therefore to test whether the maturation status of TARP
γ -8 determines surface delivery of GluA1, we transfected TARP
γ -8 WT or TTAA mutant alone or with GluA1 in HEK-293T
cells and performed surface biotinylation assays. We observed
that a large population of TARP γ -8 WT was able to reach the
surface of heterologous cells, consistent with the observation
from our confocal microscopy study (Figure 3A). However, a
smaller amount of TARP γ -8 TTAA unglycosylated mutant was
also observed on the plasma membrane. Although there was
some surface expression, the amount was markedly decreased.
When co-expressed with GluA1, both TARP γ -8 WT and TTAA
reached the surface plasma membrane of HEK-293T cells, similar
to the single transfection of TARPs. We found that the relative
surface expression of TARP γ -8 TTAA mutant was reduced more
than 50 % compared with the surface expression of TARP γ -
8 WT (Figure 3B), by measuring the band intensity ratio of
surface-expressed TARP γ -8 WT and TTAA mutant to total
bond intensity. Interestingly, the surface delivery of GluA1 was
markedly reduced (∼40 %) when co-expressed with TARP γ -8
TTAA mutant. This finding indicates that the N-glycosylation of

c The Authors Journal compilation 
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Figure 3 Unglycosylated TARP γ -8 decreases the surface expression of
GluA1 in heterologous cells
(A) TARP γ -8 WT or TTAA mutant was co-transfected with control vector or GluA1 in
HEK-293T cells. The cells were biotinylated with a membrane-impermeant biotin and then
membrane proteins were isolated as described in the Materials and methods section. The
surface-expressed fraction was pulled down using NeutrAvidin–agarose resin, eluted with 6×
Laemmli sample buffer, resolved by SDS/PAGE, transferred on to a PVDF membrane, and
probed with anti-GluA1 or anti-TARP γ -8 antibody. (B) The amount of surface-expressed GluA1
or TARP γ -8 relative to total expression was quantified from three independent experiments.
The band intensities of total input or surface-expressed proteins were measured using ImageJ
software and the surface-expressed intensity relative to total was calculated. The value for the
TARP γ -8 TTAA condition was normalized to TARP γ -8 WT and is presented as a ratio. Left:
quantification of surface-expressed GluA1. Right: quantification of surface-expressed TARP
γ -8 when co-expressed with GluA1. Results are mean + − S.D. surface expression (GluA1,
0.60 + +
− 0.060; n = 3; **P < 0.01; γ -8, 0.44 − 0.235; n = 3; *P < 0.05, Student’s t test).
TARP γ -8 from the ER to the plasma membrane is required for
efficient GluA1 surface expression.
Total and surface expression of GluA1 is not rescued by
unglycosylated TARP γ -8 in primary hippocampal neurons
To examine the effect of maturation of TARP γ -8 on AMPA
receptor trafficking in neurons, we first knocked down the
endogenous expression of TARP γ -8 in primary hippocampal
neurons using an H1 promoter-driven lentiviral system [27]
(Figure 4A). To simultaneously rescue TARP γ -8 expression,
shRNA-resistant TARP γ -8 WT or TTAA cDNA was introduced
behind an Ub promoter with an IRES (internal ribosome entry
site)–EGFP sequence on the same shRNA lentiviral construct
(Figure 4A). The TARP γ -8 shRNA was highly effective in
knocking down the endogenous TARP γ -8 expression 10 days
after infection of primary hippocampal neurons (arrow in
Figure 4B). Total protein expression of GluA1 was also drastically
reduced (∼40 %) in TARP γ -8-knockdown neurons, consistent
with the observation from TARP γ -8-knockout hippocampus
[8,14,15,19,23,29]. Surface expression of GluA1 was also
markedly reduced by TARP γ -8 knockdown (Figures 4B and 4C),
suggesting that TARP γ -8 is required for both GluA1 trafficking
 TARP γ -8 regulates AMPA receptor trafficking 475

Figure 4 Surface and total expression of GluA1 is not rescued by unglycosylated TARP γ -8 in primary hippocampal neurons
(A) Schematic diagram of lentiviral vector for TARP γ -8 rescue expression. Endogenous TARP γ -8 was knocked down by shRNA under the H1 promoter (pH1), and TARP γ -8 WT or γ -8 TTAA with
IRES–EGFP was expressed under an Ub promoter (pUb). (B) Primary hippocampal neurons were infected with the indicated lentiviruses for 10 days. The surface biotinylation assay and Western
blotting were performed as described in the Materials and methods section. The expression of TARP γ -8 WT or γ -8 TTAA is indicated with an arrow and an arrowhead respectively. Molecular masses
are indicated in kDa. (C) The amount of total GluA1 (black bar) and surface-expressed GluA1 relative to total expression (grey bar) was quantified. The band intensities were measured using ImageJ
software, normalized to control value and are presented as a ratio. Results are mean + + + +
− S.D. GluA1 expression (total γ -8 KD, 0.63 − 0.139; total γ -8 WT, 1.27 − 0.207; total γ -8 TTAA, 0.78 − 0.107;
surface γ -8 KD, 0.51 +− 0.232; surface γ -8 WT, 1.08 + 0.209; surface γ -8 TTAA, 0.62 + 0.218; n = 5; **P < 0.01, *P < 0.05, n.s., P > 0.05, Student’s t test). (D) The relative surface expression
− −
of TARP γ -8 WT compared with that of TARP γ -8 TTAA was quantified. The band intensities were measured using ImageJ software, and the ratio of surface to 10 % total is presented. Results are
mean + − S.D. TARP γ -8 expression (γ -8 WT, 1.56 + 0.093; γ -8 TTAA, 1.07 + 0.245; n = 5; **P < 0.01, Student’s t test). (E) Primary hippocampal neurons were transfected with TARP γ -8 WT
− −
or TTAA rescue constructs, and surface and total GluA1 expression was visualized with anti-GluA1 antibody. The right-hand panels show higher-magnification images of the boxed dendritic region
in the left panels. Scale bar, 20 μm. (F) Summary of quantification of surface (left-hand panel) or total (right-hand panel) expression of GluA1 in hippocampal neurons. The fluorescent signal was
measured using MetaMorph software. Results are means + + + + +
− S.D. (γ -8 WT, 1.00 − 0.07; γ -8 TTAA, 0.65 − 0.04 in surface GluA1; γ -8 WT, 1.00 − 0.12; γ -8 TTAA, 0.61 − 0.05 in total GluA1; n =
3, total neuron number >5–15, *P < 0.01, Student’s t test). KD, knockdown.

and stability. Total and surface expression of GluA1 level was
completely rescued by the lentivirus-mediated overexpression
of TARP γ -8 WT; however, it was not rescued by TARP γ -8
TTAA unglycosylated mutant (Figures 4B and 4C). The rescued
expression with the TTAA mutant is lower than with the WT,
which may be degraded through the protein-degradation pathway,
but is still higher than the original endogenous levels. Both
endogenous and overexpressed TARP γ -8 efficiently trafficked
to the neuronal surface. Notably, although TARP γ -8 TTAA was
not glycosylated at all, it was also able to traffic to the neuronal
surface. However, the efficiency of surface delivery was much
less than that of the WT (Figure 4D), suggesting that much of
surface-expressed unglycosylated TARP γ -8 is mistargeted.
To examine the role of TARP γ -8 glycosylation in surface
trafficking of endogenous GluA1, we expressed TARP γ -8
WT or TARP γ -8 TTAA in primary hippocampal neurons,
and stained the neurons with anti-GluA1 antibody recognizing
the extracellular N-terminal domain, so that surface-expressed
receptor could be specifically labelled (Figure 4E). Total GluA1
was also evaluated following permeabilization of transfected
neurons (Figure 4E). Expression of both surface and total GluA1
was markedly reduced, by ∼35 % and ∼40 % respectively,
consistent with the surface biotinylation assay, which revealed
the reduced surface expression of GluA1 when co-expressed with
TARP γ -8 TTAA.
The maturation of GluA1 is impaired in neurons expressing
unglycosylated TARP γ -8
Since the surface expression of GluA1 is impaired by
unglycosylated TARP γ -8, we expected that trafficking and


c The Authors Journal compilation 
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476 C.-Y. Zheng and others
Figure 5 Glycosylation of GluA1 is impaired in TARP γ -8-knockdown
neurons and not rescued by the unglycosylated TARP γ -8 mutant
(A) Glycosylation analysis of GluA1. GluA1 was immunoprecipitated from the membrane fraction
of primary hippocampal neurons infected with the indicated lentiviruses, and treated with Endo Hf
or PNGase F overnight at 37 ◦ C. The gel migration pattern was analysed by Western blotting using
anti-GluA1 antibody. Arrows indicate the Endo Hf-resistant ‘mature’ population, and arrowheads
represent the Endo Hf-sensitive ‘immature’ population in the Endo Hf-treated lanes. Molecular
masses are indicated in kDa. (B) The upper and lower band intensities in the Endo Hf-treated
lanes were measured using ImageJ software, and the percentage of upper band intensity to total
(upper + lower) was calculated. Results are mean + − S.D. percentage quantification of mature
GluA1 (upper band) (control, 0.82 + + +
− 0.056; γ -8 KD, 0.56 − 0.022; γ -8 WT, 0.79 − 0.007; γ -8
TTAA, 0.65 +
− 0.046, n = 3; *P < 0.05, n.s., P > 0.05, Student’s t test). KD, knockdown.
glycosylation of GluA1 from the ER–Golgi compartment to
plasma membrane is also regulated by TARP γ -8 maturation.
To evaluate the glycosylation status of GluA1 by TARP γ -8, we
used Endo Hf, which digests immature high mannose sugars, or
PNGase F, which removes all N-linked sugars. More than 80 %
of endogenous GluA1 was resistant to Endo Hf treatment, which
represents a mature population (arrow in Figure 5A). GluA1 was
more sensitive to Endo Hf treatment in TARP γ -8-knockdown
neurons (arrowhead in Figure 5A), demonstrating that TARP γ -
8 is required for GluA1 maturation. The rescue expression of
TARP γ -8 WT restored the Endo Hf sensitivity to the endogenous
control level; however, the expression of unglycosylated TARP γ -
8 TTAA mutant was not able to rescue the Endo Hf sensitivity
to the control level (Figure 5). These results indicate that the
maturation of GluA1 is impaired by unglycosylated TARP γ -8
resulting in the reduction of surface-expressed GluA1 in neurons,
thus TARP γ -8 plays a critical role in the forward trafficking of
GluA1.
DISCUSSION
Auxiliary subunits of receptors have been defined as non-pore-
forming proteins that interact directly with a channel subunit,
modulate channel properties and promote surface trafficking
together with the channel subunit in vivo [30]. Several AMPA
receptor-interacting proteins such as CNIH-2/3, CKAMP44
(cytosine-knot AMPA receptor-modulating protein 44), SynDIG1
(synapse differentiation-inducing 1), SOL-1 (suppressor of
lurcher protein 1) and GSG1L (germ cell-associated 1-lite protein)

c The Authors Journal compilation 
c 2015 Biochemical Society
have been proposed as auxiliary subunits since the TARP auxiliary
subunits were first identified [18,31–34]. In the present study,
we examined the effect of TARP γ -8 glycosylation on AMPA
receptor trafficking. We first identified glycosylation sites on
TARP γ -8 and compared glycosylation of TARP γ -8 to TARP
γ -2 and CNIH, all of which are transmembrane auxiliary subunits
of AMPA receptors. We then demonstrated that the maturation of
TARP γ -8 is required for efficient surface expression of GluA1.
Furthermore, a large pool of immature GluA1 is retained in the
ER or cis-Golgi if the N-glycosylation process of TARP γ -8 is
impaired.
N-linked oligosaccharides can be eliminated by replacing the
asparagine residue, to which oligosaccharides are covalently
attached, with glutamine. Alternatively, the glycosylation site
can be disrupted by mutating the serine or threonine within
the consensus motif to an alanine. In our study, we generated
the glycosylation mutants by replacing a conserved threonine
residue of TARPs with an alanine residue (Figure 1). An
alternative chemical approach to analyse the effects of N-
glycosylation is treatment with tunicamycin, which blocks the
maturation of N-glycans by inhibiting N-acetylglucosamine
transferases [35]. We did not utilize tunicamycin because AMPA
receptors also contain four to six consensus sequences of N-
glycosylation [36], and therefore would also be affected by
tunicamycin treatment. To avoid disrupting AMPA receptor
glycosylation, we focused on point mutations in TARP γ -8.
In general, ER exit is a tightly regulated checkpoint in the
intracellular trafficking of surface-expressed or secretory proteins.
A failure of the proper trimming and branching of glycoproteins
causes the proteins to be retained in the ER and impedes further
trafficking, resulting in protein degradation or aggregation [37].
A previous study showed that TARP γ -2 N48Q unglycosylated
mutant is not trafficked to the plasma membrane, but distributed
in a punctate pattern within the cytoplasm of fibroblasts [28].
Similarly, we found that a substantial amount of unglycosylated
TARP γ -8 is unable to be exported to the plasma membrane both
in heterologous cells and in primary neurons (Figures 4 and 5). In
addition, our confocal experiments showed that unglycosylated
TARP γ -8 is accumulated in intracellular compartments.
The surface expression of GluA1 requires TARPs as essential
auxiliary subunits. Similar to the findings from TARP γ -2-
knockout mice [22], we found that the surface expression and
maintenance of AMPA receptors are markedly diminished in
TARP γ -8-knockdown primary hippocampal neurons (Figures 4
and 5). Of particular interest, total GluA1 expression was
markedly reduced upon TARP γ -8 knockdown in our study.
This finding is consistent with the observations from TARP
γ -8-knockout hippocampi, in which the total protein level of
AMPA receptors is severely diminished [14,15,19,23]. This
result indicates that an essential role of TARP γ -8 is probably
the stabilization of the assembly of AMPA receptor complex.
In addition, the overexpression of unglycosylated γ -8 did not
rescue the surface and total expression of GluA1 compared with
TARP γ -8 WT in neurons (Figure 4), indicating that TARP γ -
8 maturation is required for the surface trafficking of AMPA
receptors. In addition, TARP γ -8 maturation is necessary for
maturation of AMPA receptors as the glycosylation status of
GluA1 was immature upon the expression of unglycosylated
TARP γ -8 (Figure 5).
In conclusion, the present study demonstrates the pivotal role of
TARP γ -8 in maintaining the stability, maturation and trafficking
of AMPA receptors in hippocampal neurons. Further study on the
role of TARPs and CNIH-2 in the detailed processes of AMPA
receptor assembly and export in a spatiotemporal manner will be
invaluable.

AUTHOR CONTRIBUTION
Chan-Ying Zheng, Young Ho Suh and Katherine Roche designed the experiments and
wrote the manuscript. Chan-Ying Zheng, Young Ho Suh and Katherine Roche performed
the experiments and analysed data. Katherine Roche supervised the project.
ACKNOWLEDGEMENTS
We thank J.D. Badger II for technical assistance.
FUNDING
This work was supported by the National Institute of Neurological Disorders and Stroke
Intramural Research Program. Y.H.S. was supported by the Basic Science Research
Program [grant number NRF-2011-0011694] through the National Research Foundation
of Korea.
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c The Authors Journal compilation 
c 2015 Biochemical Society