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INTRODUCTION |
-Aminobutyric acid, type A
(GABAA)1
receptors are the major sites of fast synaptic inhibition in the brain.
In mammals, they are constructed as pentameric structures from multiple
subunits selected predominantly from the following distinct classes:
(1-6),
(1-3),
(1-3),
,
,
, and
, creating an
incredible (165) potential for structural diversity.
However, relatively few functionally distinct receptor compositions are
thought to exist in vivo (1). It is possible that multiple
receptor types may exist that are functionally equivalent. Their
distinct subunit compositions may provide subtle functions such as
modulation by endogenous ligands such as neurosteroids (2) or second
messenger systems (3, 4), subcellular localization (5), or long term
differences in the regulation of receptor surface expression (6, 7).
Despite these caveats, GABAA receptor heterogeneity occurs
via defined pathways to limit receptor diversity (3, 6). Possible
mechanisms include brain region-specific (8) and temporal expression
(9). However, many neuron types often express multiple receptor subunit
mRNAs simultaneously (8), suggesting that subcellular mechanisms
for differential receptor assembly may also exist. Potential processes
could include the discrete sites of subcellular receptor assembly (10,
11) and/or the presence of assembly signals capable of differential
interaction with other subunits.
In support of the existence of differential assembly signals,
GABAA receptor assembly appears to be strictly controlled,
producing receptors with a fixed stoichiometry of 2
, 2
, and 1
(12-17). Furthermore, GABAA receptor assembly signals have
been identified in the
1 (18, 19),
2/3 (19, 20), and
3 (21)
subunits. Although the regions identified in these studies may exhibit
subunit class-specific interactions, to date no studies have
investigated the ability of GABAA receptors to discriminate
between subunits of the same class.
Consistent with the location of these assembly signals to intersubunit
contact points, the
/
signals (18, 19, 21) are located proximal
to the GABA and benzodiazepine binding sites (22, 23) formed at subunit
interfaces between the
-
and 
subunits, respectively, and
also the
2 high affinity GABA site (24). Similarly, the homologous
region in
1 is an important component of the GABA binding domain
(25).
Given the high degree of homology between the
and
subunits in
this region combined with their differential ability to assemble with
subunits (
1 with
1-3,
2 with
3, possibly
1, but not
2) (20), we sought to investigate the role of these sequences in the
differential assembly of
/
subunits with
subunits. Using
site-directed mutagenesis, we have determined that the conversion of a
single amino acid in
1 to that of
2 (R66A) is sufficient to alter
the assembly profile of the
1 subunit to that of the
2, as
determined by immunofluorescence and cell surface ELISA. These results
demonstrate that the identity of a single amino acid may be critical in
determining assembly with particular receptor subunits and may identify
a basis for subunit-specific GABAA receptor assembly.
Furthermore, we present evidence for the existence of alternative
assembly signals, which may permit the formation of diverse receptor
types, dependent upon subunit availability.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
COS 7 cells (ATCC CRL 1651)
were maintained in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal bovine serum, 2 mM glutamine,
1 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 units/ml penicillin in an atmosphere of 5% CO2.
Exponentially growing cells were transfected by electroporation (400 V,
infinity resistance, 125 microfarads, Bio-Rad Gene Electropulser II).
10 µg of DNA was used per transfection (2 × 106
cells) using equimolar ratios of expression constructs. Cells were
analyzed 12-48 h after transfection.
DNA Constructions--
Murine
1,
1-3, and
2L subunit
cDNAs containing either the Myc or FLAG epitope tags (between amino
acids 4 and 5 of the mature polypeptide) have been described previously
and shown to be functionally silent with respect to receptor
pharmacology and physiology (5, 18, 20, 26). The mutant constructs
1S,
1(
1),
2GKER, and
3DNTK were
generated by site-directed mutagenesis as reported previously (18, 20).
The remaining mutant
1/
2 constructs were generated by
site-directed mutagenesis using the oligonucleotides:
1(
2),
5'-CATCCTTCCAAGTTTGAGCGAAAAAAATATCTATTGTATACTC-3';
1(R-A), 5'-CTTCCAACTTTGAGCGAAAAACACATC-3';
2(
1),
5'-TGTCATACCAGGATTGGCGAAAAAAAACATCAATTGTATATTC-3',
2(A-R), 5'-CATACCAGGTTTGGCGAAAAAAAATATC-3'. The
fidelity of the final expression constructs was verified by DNA sequencing.
Antibodies--
The 9E10 antibody was obtained from 9E10
hybridoma cells (27) and used directly as supernatant without
purification. The secondary antibodies, goat anti-mouse Alexa Fluor 568 and goat anti-mouse Alexa Fluor 488, were purchased from Molecular
Probes and goat anti-mouse horseradish peroxide was from
Amersham Biosciences.
Immunofluorescence--
COS7 cells were fixed in 3%
paraformaldehyde (in PBS), washed twice in 50 mM
NH4Cl (in PBS), and blocked (10% fetal bovine serum, 0.5%
bovine serum albumin in PBS) for 30 min. Subsequent washes and antibody
dilutions were performed in PBS containing 10% fetal bovine serum and
0.5% bovine serum albumin. After surface immunofluorescence cells were
permeabilized by the addition of 0.5% Triton X-100 (10 min), and the
immunofluorescence protocol was repeated from the NH4Cl
step. Cells were examined using a confocal microscope (Zeiss LSM510).
Quantification of Cell Surface Expression--
COS7 cells were
plated onto 3-cm wells of a 6-well dish. Two transfections were pooled
and used to seed 6 wells ("surface" and "total" in triplicate).
Cells were fixed in 3% paraformaldehyde (in PBS). Cell surface
detection was performed in the absence of detergent, and total
expression levels were determined following Triton X-100 (0.5%, 15 min) treatment. Cells were washed twice in 50 mM
NH4Cl (in PBS) and blocked (5% fat-free powdered milk (Marvel), 10% fetal bovine serum , 0.5% bovine serum albumin in PBS)
for 1 h. Subsequent washes were performed in block. Receptor expression was determined using a horseradish peroxide-conjugated secondary antibody and assayed using
3,3',5,5'-tetramethylbenzidine (Sigma) as the substrate, with
detection at 450 nm after 30 min, after the addition of 0.5 M H2SO4. The reaction rate was
determined to remain linear for up to 1 h (results not shown).
Immunoprecipitation--
Cells were
L-methionine-starved for 30 min before labeling with
[35S]methionine (0.5 mCi/10-cm dish,
Translabel ICN/Flow) for 4 h. Cells were lysed in 10 mM sodium phosphate buffer containing 5 mM
EDTA, 5 mM EGTA, 50 mM sodium fluoride, 50 mM sodium chloride, 1 mM sodium orthovanadate,
5 mM sodium pyrophosphate, 0.1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
antipain, 10 µg/ml pepstatin, 0.1 mg/ml aprotinin, and 2% Triton
X-100 (lysis buffer). Post-nuclear supernatants were preabsorbed with
protein A-Sepharose and immunoprecipitated with 200 µl of 9E10
supernatant in the presence of protein A-Sepharose. Pellets were washed
in lysis buffer (containing 0.5% deoxycholate and 0.2% SDS) and
centrifuged through a 30% sucrose cushion followed by 3 additional
washes in buffer supplemented with 0.5 M NaCl and a final
wash without NaCl. Pellets were then resuspended in reducing sample
buffer (2% SDS, 5%
-mercaptoethanol in 0.68 M Tris, pH
6.8) and analyzed by 8% SDS-polyacrylamide gel electrophoresis and autoradiography.
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RESULTS |
Previous studies have revealed that the subunits of the most
commonly expressed GABAA receptor (
1
2
2) cannot
reach the cell surface when expressed alone nor when
1
2L or
2
2L combinations are co-expressed. Only when
1
2 or
1
2
2L subunits are co-expressed are functional cell surface
receptors produced (5). Non-productive subunit monomers or
1
2L or
2
2L dimers are retained in the endoplasmic reticulum (ER) by
interactions with BiP or calnexin.
Receptor Homology within the Region of a Putative Assembly
Signal--
Recently, a putative assembly signal within the
1 and
6 subunits (see
6S, Fig.
1A) was identified as being
required for assembly and cell surface expression with
3 (18). This
region exhibits homology between all the GABAA receptor
subunits (Fig. 1). The highest homology exists within subunit classes
(
values = 63.2%,
values = 78.9%, and
values = 71.1%), whereas homology between the subunit classes
(including
and
) is 26.3%. Within this region, both the
glutamine (Gln-67) and the tryptophan (Trp-69) have been shown to be
essential for assembly (18, 28). However, the glutamine is completely
conserved among all GABAA receptors, and the tryptophan is
completely conserved among all members of the ligand-gated ion channel
superfamily, indicating a general role in subunit architecture or
folding.

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Fig. 1.
Sequence alignment of GABAA
receptor subunits between amino acids 57 and 94 ( 1 mouse numbering). Sequence alignments
over this region of (A), (B), and (C) subunits are shown. Consensus sequences are shown for
each individual subunit class. An overall consensus sequence is shown
for , , and subunits (D) along with the sequences
of and subunits. The amino acid underlined (in
bold) identifies a single residue completely conserved
within each subunit class but absent from all other subunits.
Completely conserved tryptophans (in all members of the ligand-gated
superfamily) are illustrated in bold.
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To implicate residues that may be involved in subunit class-specific
assembly of GABAA receptors, we examined the sequences in
this region to identify residues completely conserved within a subunit
class but absent from the other subunit classes. Surprisingly, despite
the high degree of homology evident, only one residue (Arg-66 in
1)
fits this criterion. At this position, an arginine (
s), glutamine
(
s), or alanine (
s), is always present (histidine in
, serine
in
). Interestingly, this residue in
1 has been shown to be
critical for GABA binding (22).
To assess the role of this region in GABAA receptor
assembly we investigated
1/
2 subunit chimeras and mutants for
their ability to assemble with the
subunits. The constructs used
are
1myc,
1Smyc (lacking residues
57-68),
1(
2)myc (residues 57-68 replaced with
the homologous residues from
2),
1(R-A)myc
(arginine at position 66 mutated to alanine),
2Lmyc,
2L(
1)myc (residues replaced with homologous residues
from
1), and
2L(A-R)myc. Subunit detection was
performed using 9E10 antibodies that recognize the Myc epitope present
in all the
1/
2L subunits. All
subunits used were
epitope-tagged with the FLAG epitope. When expressed alone, all the
above
1/
2L subunits do not reach the cell surface but are
retained within the ER (5) (results not shown). Thus, we measure the
ability of the
1/
2L subunits to be rescued from the ER and
expressed on the surface as heteromeric receptors with
subunits.
Cell Surface Expression of
1/
2 Subunits with
2--
COS7 cells were transfected with
/
myc
2FLAG, and immunofluorescence was
performed using 9E10 antibodies. As shown previously (5, 26),
1 and
2 subunits are able to assemble and form heteromeric receptors on
the cell surface (Fig. 2A).
Consistent with the presence of an assembly signal existing between
residues 57 and 68 of the
1 subunit,
1S (lacking this region)
cannot reach the cell surface despite the presence of the
2 subunit.
The
1S is retained within the ER, as evidenced by the classic
reticular staining pattern observed (Fig. 2A). To address
the possibility that this subunit might misfold, we examined an
1(
1) chimera in which residues 57-68 in the
1
were replaced with the homologous residues from GABAC
receptor
1 subunit (18). Given the expected structural similarities
between
1 and GABAA receptor subunits combined with the
recent observation that this region in
1 also contributes to the
GABA binding site (25), this chimera would be less likely to misfold.
In keeping with the inability of the
1 subunit to assemble with the
GABAA receptor
subunits (29, 30), coexpression of
1(
1) with
2 did not lead to cell surface
expression (results not shown).

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Fig. 2.
Surface expression of
2 recombinant GABAA receptors requires
the presence of the 1 subunit. COS 7 cells coexpressing 2FLAG with either
1myc, 1Smyc, 2Lmyc,
1( 2)myc, 1(R-A)myc,
2L( 1)myc, or 2(A-R)myc were examined
by immunofluorescence (A) for the presence of the Myc-tagged
subunits at the surface and intracellularly. Quantification of surface
expression levels (B) were performed by cell ELISA in the
absence (Surface) or presence (total) of detergent
and normalized to 1 levels. COS 7 cells were coexpressing
2FLAG with either 1myc (lane
1), 1Smyc (lane 2), 2Lmyc
(lane 3), 1( 2)myc (lane 4),
1(R-A)myc (lane 5),
2L( 1)myc (lane 6), or
2(A-R)myc (lane 7). Each recording represents
the mean ± S.E. of at least nine determinants in at least three
independent experiments. Results are significantly different
from 1 control (p < 0.001, t
test).
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Receptors composed of
2
2 do not access the cell surface but are
retained in the ER (5) (Fig. 2). Therefore, we assessed the ability of
1(
2) to assemble with
2. Consistent with a role
for
1 residues 57-68 in assembly with
2, cell surface expression
of
1(
2) is abolished.
Given the critical role of arginine at position 66 of
1 in GABA
binding (22) and the complete conservation of this position within all
subunit classes (Fig. 1), we mutated this arginine (in
1) to the
corresponding residue (alanine) in
2 creating
1(R-A).
This mutation completely abolished the ability of
1 to assemble with
2.
To determine whether this putative
1 assembly signal might be
transplanted into the
2 subunit, we generated a
2L(
1) construct containing the residues 57-68 from
1 in place of the homologous
2 sequence. When expressed with
2,
2L(
1) is still incapable of assembling with
2 (Fig. 2A). Thus, although the
1 region 57-68 is
essential for the assembly of
1 and
2 and critically dependent
upon an arginine at position 66, this region is insufficient to direct
this assembly event. This is corroborated by the
2L(A-R)
construct, which cannot assemble with
2.
Quantification of these observations was performed by whole cell ELISA
to determine receptor cell surface expression (no detergent) versus total (detergent) receptor expression. The values
presented here do not reflect a true percentage of surface-expressed
receptors for two reasons; first, the
1
2 values have been
normalized to 100% and, second, surface values greater than 100% have
been detected for
3 homomers (results not shown), suggesting a
possible inhibitory influence of prior detergent treatment on this
assay. In these experiments (Fig. 2B, Table
I) it can be seen clearly that the assembly of
2 with
1 requires residues 57-68 of
1 (
1S = 0.9 ± 1.3%), most notably an arginine at position 66 (
1(R-A) = 5.0 ± 4.3%). In agreement with the
results observed by immunofluorescence, although this region is
critically involved in assembly with
2, transplantation of this
signal to
2 does not confer the ability to assemble with
2
(
1(
2) = 3.7 ± 3.1%).
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Table I
Summary of cell surface expression for mutant GABAA receptors
Values are obtained from Fig. 2-6 and are expressed as the percentage
cell surface expression relative to 1 (normalized to 100%) ± S.E.
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Cell Surface Expression of
1/
2 Subunits with
1--
COS7 cells were transfected with
/
myc
1FLAG, and immunofluorescence was
performed using 9E10 antibodies. As shown previously (26)
1 and
1
subunits are able to assemble and form heteromeric receptors on the
cell surface (Fig. 3). Consistent with
the presence of an assembly signal existing between residues 57-68 of
the
1 subunit,
1S cannot reach the cell surface despite the
presence of the
1 subunit (2.7 ± 3.1%). Similar results were
observed for the
1(
1) (results not shown).

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Fig. 3.
Surface expression of
1 recombinant GABAA receptors requires
the presence of the 1 subunit. COS 7 cells coexpressing 1FLAG with either
1myc, 1Smyc, 2Lmyc,
1( 2)myc, 1(R-A)myc,
2L( 1)myc, or 2(A-R)myc were examined
by immunofluorescence (A) for the presence of the Myc-tagged
subunits at the surface and intracellularly. Quantification of surface
expression levels (B) were performed by cell ELISA in the
absence (Surface) or presence (total) of detergent
and normalized to 1 levels. COS 7 cells were coexpressing
1FLAG with either 1myc (lane
1), 1Smyc (lane 2), 2Lmyc
(lane 3), 1( 2)myc (lane 4),
1(R-A)myc (lane 5),
2L( 1)myc (lane 6), or
2(A-R)myc (lane 7). Each recording represents
the mean ± S.E. of at least nine determinants in at least three
independent experiments. *, denotes significant difference from 1
control (p < 0.001, t test).
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In this study, we could detect only very low levels of
2L
1
surface receptors by immunofluorescence (Fig. 3A) that could not be resolved from background (mock-transfected cells) when analyzed
quantitatively (10.2 ± 11.8%, Fig. 3B). To determine whether the robust surface expression observed upon
1
1 expression results from the use of the
1 57-68 assembly signal, we assessed the ability of
1(
2) to assemble with
1. In
contrast to the results observed with the
2 subunit, cell surface
expression of
1(
2) is not significantly different
(108.6 ± 23.4%) from that observed for wild-type
1 (100 ± 7.6%) but higher than that observed for the wild-type
2L
(10.2 ± 11.8%). These findings suggest that, in contrast to the
2 subunit, the
1 subunit does not exhibit the same requirements
for this region of the
1 for the assembly of
1
1 heteromeric
receptors. This possibility is corroborated by the efficient assembly
of
1 with
1(R-A) (83.9 ± 16.1%, Fig. 3).
However, this region is still essential for
1
1 receptor assembly,
as evidenced by the inability of
1S (Fig. 3) and
1(
1) (results not shown) to assemble with
1. Both
of the
2 mutants (
2(
1) and
2(A-R))
were indistinguishable from the wild-type
2L with respect to cell
surface expression with
1, i.e. weak surface
immunofluorescence and non-detectable surface levels by ELISA (Fig.
3).
Cell Surface Expression of
1/
2 Subunits with
3--
COS7 cells were transfected with
/
myc
3FLAG, and immunofluorescence was
performed using 9E10 antibodies. As shown previously (26)
1 and
3
subunits are able to assemble and form heteromeric receptors on the
cell surface (Fig. 3). Consistent with the presence of an assembly
signal existing between residues 57 and 68 of the
1 subunit,
1S
cannot reach the cell surface (3.5 ± 3.6%) despite the presence
of the
3 subunit. Similar results were observed for the
1(
1) (results not shown).
Receptors composed of
3
2 have been reported to be capable
of forming functional cell surface receptors (18). In this study, we
observed robust surface expression by immunofluorescence (Fig. 4A) and ELISA (54.7 ± 19.7%, Fig. 4B). We assessed the ability of
1(
2) to assemble with
3. As observed with the
1
subunit,
1(
2) is capable of assembling with
3 and
even exhibits enhanced levels of surface expression (163 ± 21.9%, p < 0.02, t test) compared with
1 (100 ± 10.5%) or
2L (54.7 ± 19.7%). These
findings suggest that, like the
1 subunit but in contrast to the
2 subunit,
3 does not require this region of the
1 for the
assembly of
1
3 heteromeric receptors. This possibility is
corroborated by the efficient assembly of
3 with
1(R-A) (81.2 ± 9.2%, Fig. 4). Both of the
2
mutants (
2(
1) and
2(A-R)) were
indistinguishable from the wild-type
2L with respect to cell surface
expression with
3, i.e. strong surface immunofluorescence and detectable surface levels by ELISA (Fig. 4,
2L(
1) = 50.9 ± 16.2% and
2L(A-R) = 85 ± 23.3%).

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Fig. 4.
Surface expression of
3 recombinant GABAA receptors requires
the presence of the 1 subunit. COS 7 cells coexpressing 3FLAG with either
1myc, 1Smyc, 2Lmyc,
1( 2)myc, 1(R-A)myc,
2L( 1)myc, or 2(A-R)myc were examined
by immunofluorescence (A) for the presence of the Myc-tagged
subunits at the surface and intracellularly. Quantification of surface
expression levels (B) were performed by cell ELISA in the
absence (Surface) or presence (total) of detergent
and normalized to 1 levels. COS 7 cells coexpress
3FLAG with either 1myc (lane
1), 1Smyc (lane 2), 2Lmyc
(lane 3), 1( 2)myc (lane 4),
1(R-A)myc (lane 5),
2L( 1)myc (lane 6) or
2(A-R)myc (lane 7). Each recording represents
the mean ± S.E. of at least nine determinants in at least three
independent experiments. *, denotes significant difference from 1
control (*, p < 0.001; **, p < 0.05 t test).
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Interestingly, the
3 subunit (20, 31) and to a lesser extent the
1 subunit (32, 33) can form functional homomeric ion channels (not
GABA-gated), whereas the
2 subunit is incapable of exiting the ER
(5). A four-amino acid signal (GKER) that controls
3
homooligomerization has been identified (20). When transferred to
2
(
2GKER), this subunit is capable of cell surface
expression as functional ion channels. The reciprocal construct
(
3DNTK) can no longer assemble into homomeric receptors
but is retained in the ER (20). To determine whether the assembly
signal identified in the
1 may also recognize the homomeric assembly
signal present in the
3 subunit, we analyzed the assembly of
1/
2 subunits with the
2GKER and
3DNTK subunits.
Cell Surface Expression of
1/
2 Subunits with
2--
COS7 cells were transfected with
/
myc
2GKER(FLAG), and
immunofluorescence was performed using 9E10 antibodies. As expected,
1 and
2GKER subunits are able to assemble and form
heteromeric receptors on the cell surface (Fig.
5). Again,
1S cannot reach the cell surface (9.7 ± 6.9%) despite the presence of the
2GKER subunit. Similar results were observed for the
1(
1) (results not shown). In contrast to the ER
retention of
2
2L complexes (5) (3.0 ± 3.0% surface
expression, Fig. 2), when
2GKER and
2L are
coexpressed, cell surface (74.3 ± 19.9%) receptors are produced
(Fig. 5). Furthermore, surface expression with
1(
2)
was increased for
2GKER (118.6 ± 21.9%) compared
with
2 (10.2 ± 10%). More striking is the ability of the
1(R-A) to assemble with
2GKER and reach
the cell surface (90.1 ± 24.3%) compared with
2 (5.0 ± 4.3%). Analysis of the
2 mutants reveals that the
2L(
1) and the
2L(A-R) are also
expressed on the cell surface at significant levels (55.8 ± 26.0% and 31.3 ± 9.9%) comparable with those observed for
3
(50.9 ± 16.2% and 85 ± 23.3%, respectively), not
2
(3.7 ± 3.1% and 7.5 ± 6.0%, respectively). The results
with the
2GKER construct identifies the
3 homomeric
assembly signal as capable of conferring
3-like assembly
characteristics to the
2 subunit and suggests that, although
3
requires the presence of
1 signal, the homomeric assembly signal in
3 does not depend critically upon the identity of the sequence
between 57-68 of the
1 and is capable of assembling with both
1
or
2L.

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Fig. 5.
Surface expression of
2GKER recombinant GABAA
receptors requires the presence of the 1
subunit. COS 7 cells coexpressing 2GKER/FLAG with
either 1myc, 1Smyc, 2Lmyc,
1( 2)myc, 1(R-A)myc,
2L( 1)myc, or 2(A-R)myc were examined
by immunofluorescence (A) for the presence of the Myc-tagged
subunits at the surface and intracellularly. Quantification of surface
expression levels (B) was performed by cell ELISA in the
absence (Surface) or presence (total) of detergent
and normalized to 1 levels. COS 7 cells coexpressing
2GKER/FLAG with either 1myc (lane
1), 1Smyc (lane 2), 2Lmyc
(lane 3), 1( 2)myc (lane 4),
1(R-A)myc (lane 5),
2L( 1)myc (lane 6), or
2(A-R)myc (lane 7). Each recording represents
the mean ± S.E. of at least nine determinants in at least three
independent experiments. *, denotes significant difference from 1
control (p < 0.001, t test).
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Cell Surface Expression of
1/
2 Subunits with
3DNTK--
COS7 cells were transfected with
/
myc
3DNTK(FLAG), and
immunofluorescence was performed using 9E10 antibodies. As expected,
1 and
3DNTK subunits are able to assemble and form
heteromeric receptors on the cell surface (Fig.
6). Again,
1S cannot reach the cell surface (7.9 ± 5.6%) despite the presence of the
3DNTK subunit. Similar results were observed for the
1(
1) (results not shown). As observed for the
3
2L receptors (54.7 ± 19.7% surface expression, Fig. 6),
when
3DNTK and
2L are coexpressed, cell surface
(26.8 ± 11.5%) receptors are produced (Fig. 6). Furthermore,
surface expression of
1(
2) with
3DNTK
occurred at significant levels (99.6 ± 5.8%). In addition, the ability of the
1(R-A) to assemble with
3DNTK and reach the cell surface (61.6 ± 13.2%)
compared with
3 (81.2 ± 9.2%) was unaffected. Analysis of the
2 mutants reveals that the
2L(
1) and the
2L(A-R) are expressed on the cell surface at significant
levels (38.3 ± 20.5 and 24.7 ± 13.2%), comparable with
those observed for
3 (50.9 ± 16.2 and 85 ± 23.3%,
respectively) but not
2 (3.7 ± 3.1% and 7.5 ± 6.0, respectively). Although the values obtained for the assembly of
3DNTK are consistently lower than that observed for the
wild-type
3, the differences are not statistically significant, with
the exception of
1(
2) and
2(A-R)
(p < 0.005, t test). Paradoxically, results
observed for
3DNTK suggests that the
3 homomeric
assembly signal is not an essential requirement for the assembly of
3 with either
1 or
2.

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Fig. 6.
Surface expression of
3DNTK recombinant GABAA
receptors requires the presence of the 1
subunit. COS 7 cells coexpressing 3DNTK/FLAG with
either 1myc, 1Smyc, 2Lmyc,
1( 2)myc, 1(R-A)myc,
2L( 1)myc, or 2(A-R)myc were examined
by immunofluorescence (A) for the presence of the Myc-tagged
subunits at the surface and intracellularly. Quantification of surface
expression levels (B) were performed by cell ELISA in the
absence (Surface) or presence (total) of detergent
and normalized to 1 levels. COS 7 cells coexpressing
3DNTK/FLAG with either 1myc (lane
1), 1Smyc (lane 2), 2Lmyc
(lane 3), 1( 2)myc (lane 4),
1(R-A)myc (lane 5),
2L( 1)myc (lane 6), or 2(A-R)
(lane 7). Each recording represents the mean ± S.E. of
at least nine determinants in at least three independent experiments.
*, denotes significant difference from 1 control (p < 0.001, t test).
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|
To determine the ability of the
1/
2 polypeptides to oligomerize
with
2, cDNAs were cotransfected into COS7 cells,
[35S]methionine-labeled, and immunoprecipitated via the
Myc epitope tag on the
1/
2 subunits. Only the extracellular
domain of
2 was used to eliminate any contribution from other
subunit interactions (34) and events at the cell surface such as
receptor turnover (6). Bands corresponding to the
1/
2 and
2
extracellular fragments were excised and quantified using a
scintillation counter. The ratio of
2 co-immunoprecipitated with the
1/
2 subunit was normalized to that of wild-type
1 (39%) such
that the ratio (
2:
1) of
2 co-immunoprecipitated by
1
represents 100%. No significant reduction in binding was evident for
any of the
1/
2 subunits (Fig.
7). This is not surprising,
because each subunit must possess at least two interfaces (and
presumably assembly signals) with other subunits (Fig.
8).

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Fig. 7.
Oligomerization of
/ subunits with
2. COS 7 cells coexpressing the entire
N-terminal extracellular region of 2 ( 2- extFLAG)
with either 1myc, 1Smyc,
2Lmyc, 1( 2)myc,
1(R-A)myc, 2L( 1)myc, or
2(A-R)myc were labeled with
[35S]methionine, immunoprecipitated with antibodies
against the Myc epitope (9E10), separated by SDS-PAGE, and examined by
autoradiography. Bands representing the / and
2-extFLAG were excised and counted by scintillation. The
ratio of / : 2 was determined and normalized to that of
1.
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Fig. 8.
Identification and location of putative
assembly signals in GABAA receptor subunits.
A, putative assembly signals are illustrated for Interface 1 and their opposite interface (Interface 2) shown. 1B may
form an interface with either a (a) or a (b)
subunit. 3B may possess alternative signals capable of
interacting with 1A/ 2A. Two different 2A signals have been
identified. <GABA or >GABA represents the low
or high affinity GABA binding sites, respectively. 1, Ref.
18. 2, Ref. 39. 3, Ref. 20. 4, Ref.
19. 5, Ref. 21. *, this study. B, arrangement of
subunits in an   pentameric receptor indicating subunit
interfaces and binding sites for GABA (G) and
benzodiazepines (Bz). The mirror image of this structure is
equally possible (39).
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DISCUSSION |
To date 16 different GABAA receptor cDNAs have
been isolated from a variety of vertebrates (4). Many of these subunits exhibit differing patterns of both spatial and developmental expression in the CNS, with many neurons often expressing multiple numbers of
receptor subunits (8, 35). A major challenge in trying to analyze the
diversity of GABAA receptor structure in the brain is
determining what processes control receptor assembly. The determination of receptor composition may arise from temporal and spatial regulation, subcellular subunit segregation, and/or differential
assembly/stability. Although there is extensive evidence for a role of
temporal (9) and spatial (8, 35) regulation of subunit expression in
determining receptor composition, these mechanisms cannot explain how
receptor diversity is limited in neurons co-expressing multiple
GABAA receptor subunits simultaneously (8, 35). To date,
there is no supporting evidence for discrete subcellular assembly sites
for GABAA receptors, although emerging evidence for the
localized translation of proteins at synapses (11) may be relevant.
The potential for hierarchical assembly signals is supported by the
strict control of receptor stoichiometry (12-17) and the observations
that
1
3 (versus
3 homomers) and
1
2
2
(versus
1
2) receptors form to the exclusion of the
other possible combinations (3, 31, 36). In vivo evidence
for hierarchical receptor assembly has been provided by the analysis of
6 knockout mice, which determined that the
subunit was
concomitantly "knocked-out" by a partial
6 polypeptide product
that remains able to associate with the
subunit but cannot produce
functional receptors (37).
The discovery of putative GABAA receptor assembly signals
began with the identification of a natural splice variant of
6 that
lacked 10 amino acids in the N-terminal extracellular domain (38). This
splice variant (termed
6Short) was determined to be incapable of
assembling into functional receptors (18, 38). The high degree of
homology between all GABAA receptor subunits within this
region suggests a common role in receptor assembly, a possibility
vindicated for the
1 (18) (but see Ref. 19) and
3 (21). Putative
assembly signals have also been discovered adjacent to this region in
1 (for binding to
2) (39) and
2 (for binding to
1 and
3)
(19). Furthermore, two invariant tryptophans within this region have
been shown to be essential for GABAA receptor assembly and
benzodiazepine, but not GABA, binding (28). Indeed these tryptophans
are completely conserved in all members of the ligand-gated ion channel
superfamily and may provide some common structural feature necessary
for receptor assembly (28).
Given the similarity between the homologous regions between
(MEYTIDVFFRQSW) and
(MEYTIDIFFAQTW) subunits, we examined the
possibility that the sequence differences between these subunit classes
might be responsible for the differential ability of the
2 subunit
to assemble with
1 and
3 (20, 33), but not
2 (5, 20), whereas
the
1 can assemble with all three
subunits (26).
Consistent with previous findings,
1S and
1(
1)
(18) are not able to assemble with
1-3. In addition,
2L cannot
assemble with
2 (20) and only weakly with
1 (Table I) (3). In
keeping with the possibility that
1-(57-68) constitutes an assembly
signal determining oligomerization with
subunits (18),
1(
2) is also unable to assemble with
2. In fact, a
single site (Arg-66) was found to be critical for the assembly of
1
2 receptors. However, the failure of
2L(
1) to
assemble with
2 suggests that this putative assembly signal is
necessary but not sufficient to direct this oligomerization step.
Interestingly, a striking overlap between the
A site and the GABA
binding site (loop D) exists for
1
2 receptors (22). Boileau
et al. (22) showed that residues Phe-64, Arg-66, and Ser-68 were found to be part of (or close to) the GABA binding site,
with Phe-64 and Arg-66 critical for modulation by GABA. In keeping with
other studies (18, 28), these authors found that Gln-67 and Trp-69 were
essential for the production of functional receptors. Moreover, this
region is not only important in the
1 subunit contribution to GABA
binding, but the high affinity GABA binding site has been shown to be
produced by the homologous region in
2 (
2A interface to
1)
(24). In addition, the benzodiazepine binding site on the
2 subunit
resides within the same homologous region, with A79 (homologous
position to Arg-66 in
1) lining the benzodiazepine binding pocket
(23). A similar overlap between assembly signal and benzodiazepine
binding is observed for the opposing interface on the
1 subunit,
with residues 74-123 providing the binding site (40) and residues
81-100 (MTVLRLNNLMASKIWTPDTFF, see Fig. 1) involved in oligomerization
with
2 (39).
In contrast to the tight correlation between receptor assembly and the
formation of the GABA/benzodiazepine binding site in
1
2
2
receptors, little overlap exists for the opposing side of this
interface, the
B site. This side of the interface has been
determined to be constructed from 3 distinct regions; loop A between
residues 93 and 101, loop B between residues 157 and 160, and loop C
between residues 202 and 209 to generate the GABA binding domain on the
2 subunit (41, 42). The residues required for assembly
(GDKAVTGVER in
3) fall between loops B and C.
In contrast to the findings for the
2 subunit, although
1,
3,
2GKER, and
3DNTK all require the presence
of residues 57-68 from either
1 or
2, they do not exhibit the
same dependence upon this sequence as
2. The most likely explanation
for these results is a general requirement for this region to either
induce correct folding or subunit architecture but not to provide an
assembly signal. This would be consistent with the role of these
sequences in the formation of GABA and benzodiazepine binding sites.
Alternatively, assembly signals for
1 and
3 may reside within
this region, as for assembly with
2, but be interchangeable between
1 and
2. However, this seems unlikely given that
1 assembles
only poorly with
2L, yet robustly with
1(
2). Thus, it seems
more likely that
1 and
3 utilize unique assembly signals in
1
and
2. This is consistent with previous findings (19) in which
assembly signals just downstream from the
1 57-68 region have been
identified for
2 binding to
3 (Figs. 1 and 8). However, unlike
the
2,
3 does utilize the homologous assembly signal
(MEYQIDIFFAQTW) used by
1 (to assemble with
2) to assemble with
3 (21).
A more complex scenario is also possible. Perhaps
1 and
3, but
not
2, may possess and utilize alternative assembly signals dependent on availability. In this way GABAA receptors may
form their preferred receptor compositions depending on subunit
availability. In other words, a process of hierarchical assembly may
operate, as observed previously (3, 31, 36). Such a possibility is
supported by the observation that although "GKER" in
3 is sufficient to drive assembly with
1 and
2 (see
2GKER), it is not necessary to do so (see
3DNTK). Thus, it seems possible that multiple,
alternative assembly signals may exist for GABAA receptor
formation, providing a flexible mechanism for the construction of
GABAA receptors. In such a scenario, a
subunit would be
able to select its most desirable companion available without
committing itself to just one partner. This is observed in
knockout
mice, in which
4 and
6 subunits, normally assembling with the
subunit, are now free to assemble with
2 (35, 43, 44). Thus, the
selection of a second choice would not be expected to be possible in
the presence of a favorite. This is well illustrated by the fatal
attraction suffered for the
subunit in cerebellar granule neurons
of
6 knockout mice (37) (that retain the
6 assembly signal),
implying a complete devotion of the
subunit for
6 assembly
signal within this environment.
In summary, we have identified a single amino acid within the proposed
assembly signal of
1 for
subunits that is absolutely required
for
1
2, but not
1
1 or
1
3, receptor formation.
1,
2, and
3 exhibit distinct assembly profiles with
1 and
2, utilizing distinct assembly signals in
1/
2. We have identified an
assembly signal in
3 that is sufficient to drive assembly with both
1 and
2. However, it is not necessary, implicating the presence
of multiple assembly signals capable of fulfilling the same function,
selecting a subunit with which to assemble.