 |
INTRODUCTION |
Understanding the assembly and molecular interactions of proteins
is a rapidly growing field in biology. Many techniques have been used
to probe the interaction of proteins including, for example,
immunoprecipitation, yeast two-hybrid analysis, circular dichroism, and
more recently fluorescence energy transfer between two proteins in
close proximity (1-3). We have used the technique of fluorescence
resonance energy transfer to investigate the stoichiometric assembly of
a GABAA1
receptor, a member of the major inhibitory ligand-gated ion channel family in the brain.
It is proposed that the GABAA receptors are
hetero-oligomeric pentamers composed of
,
, and a third subunit
type,
,
, or
. This is based on an analogy with other
ligand-gated ion channels such as the nicotinic acetylcholine receptor
(4, 5), their size as measured by gel filtration and sucrose density
centrifugation (6, 7), and their image under electron microscopy (8). To date, 15 GABAA subunits have been cloned
(
1-6,
1-4,
1-3,
,
and
), and expression studies reveal that for the most characterized
subtypes at least one
, one
, and one
-subunit are required to
recover a fully functional GABAA receptor (9). We have
investigated the stoichiometry of the most abundant GABAA
receptor isoform in the brain,
1
2
2, which is responsible for at least some of the therapeutic effects of benzodiazepines, barbiturates, and steroids (10). Receptors modified with a c-Myc epitope on either the
1-,
2-, or
2- subunit were expressed transiently in HEK293 cells.
It has previously been shown that receptors transiently transfected in
these cells using the same methods as employed here are appropriately
assembled into 

-hetero-oligomers and are expressed on the cell
surface. Other subunit assemblies do not reach the cell surface and are
retained in the endoplasmic reticulum (12).
mAbs to the c-Myc epitope were derivatized with the fluorescence donor,
europium cryptate (EuK), or the fluorescent energy acceptor, XL-665.
Europium, encaged by cryptate, emits a strong long lived fluorescent
signal at 620 nm when illuminated with light at 337 nm from a nitrogen
laser, which can be time-resolved from short lived background
fluorescence. In addition to being used as a single label, analogous to
a radiolabel for example, EuK also serves as an energy donor. The
fluorescent signal produced by EuK can be transferred to an acceptor
molecule if it is in close enough proximity. The recipient molecule for
this fluorescent resonance energy transfer is a modified
allophycocyanine, XL665, which fluoresces at 665 nm (for review of the
homogeneous time-resolved fluorescence technology see Mathis (1)). The
transfer of energy from europium cryptate-labeled c-Myc antibodies to
XL-665-labeled antibodies is therefore indicative of the two antibodies
being in close proximity. Because the energy transfer is 50% at a
distance of 9.5 nm (1), this would require that the antibodies are very closely associated, for example in the same macromolecular complex.
We used EuK-labeled c-Myc mAbs to quantify the number of subunits
present on intact cells expressing
1
2
2 subunits where each of
the subunits was epitope-tagged with c-Myc and compared this with the
number of receptors present using conventional radioligand binding.
Fluorescence energy transfer was then used to confirm the stoichiometry
of the receptor as
(
1)2(
2)2(
2)1.
 |
EXPERIMENTAL PROCEDURES |
[methyl-3H]Ro15-1788 (87.0 Ci/mmol) was
from NEN Life Science Products, Hertfordshire, United Kingdom.
Flunitrazepam, GABA, and fetal calf serum were from Sigma. Minimal
essential medium (MEM) was from Life Technologies, Inc.
Construction of c-Myc Epitope-tagged GABAA Receptor
Subunits and Transient Transfections--
The GABAA
receptor
1-subunit was epitope-tagged by
site-directed mutagenesis using methods described previously (13). The epitope sequence (EQKLISEEDL) was introduced between Glu34
and Leu35 (these two residues becoming the first and last
amino acid of the epitope tag), just C-terminal to the putative signal
peptide cleavage site. The
2 and
2
GABAA receptor subunits were epitope-tagged using a
modified version of the pcDNA1Amp eukaryotic expression vector
(pcDNA1AmpSignalMyc). This vector was constructed from the
1 c-Myc cDNA described above and contains the
5'-untranslated region of bovine GABAA
1-subunit (GenBank accession no. X05717), the signal
peptide and six amino acids of the mature
1-subunit, and
the c-Myc epitope tag sequence (EQKLISEEDL (14)) followed by a small
polylinker into which the mature polypeptide of the subunit of interest
can be inserted. The human
2-subunit c-Myc construct
contains amino acids Glu38-Asn474 of
2, and the human
2-subunit c-Myc
construct contains amino acids Tyr46-le467.
All constructs were confirmed by DNA sequencing. Constructs were
prepared using polymerase chain reaction to generate the appropriate
2 and
2 sequences, which were then
subcloned into the pcDNA1AmpSignalMyc vector.
DNAs were prepared for transfection by CsCl centrifugation. Transient
transfection into HEK293 cells was performed exactly as described
previously in detail using a 1:1:1 ratio of
1,
2, and
2 cDNAs (12, 15).
Radioligand Binding--
The benzodiazepine site of the GABA
receptor was labeled by the antagonist [3H]Ro15-1788, a
radioligand frequently used to quantify receptors, because only fully
assembled 

heterotrimers bind this ligand (7, 12, 16).
Nonspecific binding was determined using 10 µM
flunitrazepam. Binding to the GABA binding site was carried out with
[3H]muscimol, and 100 µM GABA was used to
define nonspecific binding. Radioligand binding assays were carried out
in a total volume of 0.5 ml in 10 mM
KH2PO4, 100 mM KCl, pH 7.4, for
1 h at 4 °C prior to termination through Whatman GF/C filters
followed by 3× 3-ml washes with cold assay buffer and scintillation
counting. In binding studies carried out at one concentration of
[3H]Ro15-1788, this was 1.8 nM.
Immunoprecipitation--
GABAA receptors solubilized
from stably transfected cells were immunoprecipitated using
anti-
1-,
-, or
2-subunit antisera bound to protein A-Sepharose (7). 50 µl of polyclonal antiserum were
incubated with 50 µl of packed protein A beads in a total volume of 1 ml of Tris-buffered saline for 1 h at room temperature. Receptors
were solubilized from the cells using a deoxycholate/Triton buffer (1%
Triton X-100, 0.5% deoxycholate, 0.1 mM KCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride, 100 mM Tris-HCl, pH 8.2) by mixing cell membranes
for 1 h at 4 °C with a detergent buffer at a protein
concentration of 1 mg/ml.
Aliquots (500 µl) of detergent-solubilized cell membranes were
incubated with antibodies immobilized on protein A beads overnight at
4 °C. The receptor immobilized on protein A beads was washed three
times with Tris-buffered saline/0.1% Tween 20 by centrifugation and
resuspension and was finally resuspended in 0.5 ml of Tris-buffered saline, and 10-50-µl aliquots of packed beads were used for
[3H]Ro15-1788 binding.
Generation of Europium Cryptate and XL665-labeled
Antiserum--
The monoclonal anti-c-Myc mAb, 9-E10 (ATCC no.
CRL-1729, Ref. 14) was used for these experiments. An antibody was
purified from hybridoma supernatant by Cymbus Biotech, UK, and this was then derivatized with either the fluorophore, europium cryptate (at an
average ratio of 9 molecules of europium cryptate per antibody molecule), or with the fluorescence acceptor, XL665 (at a ratio of 1 molecule of XL-665 per antibody molecule), by Cis-Bio International, Marcoule, France. Labeling of c-Myc mAb with EuK at this ratio gave 418 counts of fluorescence/s/fmol of antibody.
Binding of Europium Cryptate-labeled mAbs--
Intact cells,
transiently transfected with GABAA receptors with
c-Myc-tagged subunits, were harvested by scraping, washed once by
centrifugation (20 s at 1,000 rpm in a bench top Eppendorf Microcentrifuge), and resuspended in MEM + 5% FCS.
[3H]Ro15-1788 binding (1.8 nM) was carried
out to determine receptor density on intact cells. Cells containing the
equivalent of 100 fmol of receptor were incubated with various
concentrations of europium cryptate-derivatized mAb (0.3-30
nM) in MEM + 5% FCS at room temperature, in a volume of 1 ml for 1 h on a rotamix wheel. After 3× 1-ml washes in MEM + 5%
FCS, the cells were resuspended in 190 µl of MEM + 5% FCS, and the
fluorescence signal derived from europium cryptate-labeled c-Myc mAbs
was quantified in a 96-well low fluorescence microplate on the Packard
discovery fluorescence plate reader, following the addition of 10 µl
of 1 M KF (final concentration, 50 mM KF). In
preliminary experiments the inclusion of 5% FCS was found to improve
the viability of the cells, and KF was included to stabilize europium
cryptate and prevent oxidation of unliganded cryptate.
Energy Transfer to XL665-labeled
Antibodies--
XL665-derivatized mAbs were added in excess (10 nM) to each well, and the plate was read over a 24-h time
period. Energy transfer was optimal after the receptor had been
incubated with c-Myc-XL665 for 1 h, and the signal was stable for
up to 18 h afterward.
 |
RESULTS |
Each of the
1-,
2-, and
2-subunits was engineered to express a c-Myc epitope tag
at the N terminus, and cells expressing c-Myc-tagged
-,
-, or
-subunits or untagged receptors were generated by transient
transfection. GABAA receptors were measured using two
ligands, [3H]muscimol, a radioligand for the GABA binding
site, and [3H]Ro15-1788, a radioligand for the
benzodiazepine binding site, which binds only to fully assembled
receptors that contain an
-,
-, and
-subunit (7, 9).
Homogeneity of Expressed Receptors--
In preliminary experiments
it was important to confirm that a homogeneous population of fully
assembled receptors was being expressed in the cells. This was done in
two ways. First the number of binding sites for
[3H]muscimol and [3H]Ro15-1788 was
compared. Most evidence supports the presence of two
[3H]muscimol binding sites and one
[3H]Ro15-1788 binding site in a GABA receptor monomer in
receptors immunoprecipitated from rat brain (7, 22). Therefore, if the
majority of receptors is correctly assembled in cell lines, the ratio
of the Bmax values for
[3H]muscimol binding:[3H]Ro15-1788 binding
should be 2:1. It is possible that receptors could be expressed that
are composed of
- and
-subunits only, but these would not bind
[3H]Ro15-1788, and the ratio would therefore be higher.
Saturation analysis of [3H]muscimol and
[3H]Ro15-1788 binding was carried out in cells expressing
the untagged
1-,
2-, and
2-subunits. Maximal binding of
[3H]muscimol was 2067 ± 98 fmol/mg of protein with
a Kd of 5.6 ± 0.7 nM
(n = 3). Maximal binding of [3H]Ro15-1788
was 1226 ± 21 fmol/mg of protein with a Kd of
0.7 ± 0.05 nM (n = 3). The ratio of
the GABA:benzodiazepine sites was 1.74 ± 0.11, which is in
agreement with the expression of 

heterotrimeric receptors and
no significant expression of receptors that contain only

-subunits.
Second, the ability of antibodies raised against the
1-,
2-, and
2-subunit to
immunoprecipitate all the [3H]Ro15-1788 binding sites
from a solubilized cell membrane preparation was compared. The majority
of receptors could be immunoprecipitated with antibodies raised against
the
1-,
-, or
2-subunits
(
1, 74 ± 3%;
, 81 ± 7%; and
2, 90 ± 4% of [3H]Ro15-1788 binding
sites, n = 3). This confirms that the receptors that
bind [3H]Ro15-1788 contain at least one
-, one
-,
and one
-subunit.
Expression of c-Myc-tagged GABAA Receptors--
The
number of c-Myc-tagged subunits was quantified by labeling with
anti-c-Myc mAb derivatized with europium cryptate, as illustrated in
Fig. 1 (using the subunit arrangement
proposed by Tretter et al. (11) as a model).

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Fig. 1.
Schematic representation of the experimental
design. A, the number of subunits present is quantified
using saturation binding of EuK-derivatized c-Myc. This is compared
with the number of benzodiazepine binding sites using the radioligand
[3H]Ro15-1788. B, fluorescence resonance
energy transfer occurs only when two copies of a subunit are
present.
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|
The presence of a c-Myc tag on the N terminus of the GABAA
receptor subunits did not affect the expression of the receptor. The
number of binding sites present on cells was unaffected by the addition
of the epitope tag, and saturation analysis of
[3H]Ro15-1788 binding showed that there was no major
difference in the affinity of receptors for ligand
(Kd is as follows:
1
2
2 = 1.3 nM;
1(c-Myc)
2
2 = 1.1 nM;
1
2(c-Myc)
2 = 1.4 nM;
1
2
2(c-Myc) = 1.3 nM, n = 1). The density of receptors was
unaffected by the expression of subunits with a c-Myc epitope tag,
being 1.2-3.1 pmol/mg of protein. This is in agreement with previous studies where GABAA receptors were tagged with c-Myc and
FLAG epitopes without compromising binding, function, or modulation of
the GABAA receptor (12).
Labeling of GABAA Receptors with EuK-c-Myc Monoclonal
Antibodies--
Preliminary experiments were carried out to determine
the optimal conditions for the binding of EuK-labeled c-Myc mAbs. As shown in Fig. 2B, the
fluorescence signal emitted by EuK c-Myc at 620 nm was linear with
receptor concentration. Therefore the binding of EuK-labeled c-Myc to
the receptor was linear up to at least 100 fmol of receptor/ml.
Background fluorescence from the plate and other reagents accounted for
the fluorescence in the absence of added EuK, and this was typically
800-1500 counts/s as shown in Fig. 2A. Binding of EuK c-Myc
to cells expressing untagged receptors was significant (Fig.
2A). Efforts were made to try to reduce this
nonreceptor-mediated binding by extensive washing following incubation
with EuK-c-Myc mAb and preincubation of cells with IgG to block any IgG
binding sites on the cells. Neither of these measures reduced
nonspecific binding to control cells by more than 10% and were not
routinely adopted. Instead, each experiment contained a wild type
control (i.e. receptors expressed without any c-Myc epitope
present), and nonspecific binding to these cells was subtracted from
the total fluorescence signal, which was analogous to the methodology
employed for defining nonspecific binding in a radioligand binding
assay.

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Fig. 2.
The EuK signal from
1(c-Myc) 2
cells is specific and linear with receptor density. Binding of EuK
c-Myc was carried out at 3 nM antibody, and following
1 h of incubation with antibody, cells were washed twice before
fluorescence at 620 nm was measured. A, binding to cells
expressing the 1(c-Myc) 2 2
subtype is shown. Data shown are from two to six experiments with 100 fmol of receptors ([3H]Ro15-1788 binding sites/well).
B, linear relationship between fluorescence counts and
receptor concentration is shown. Data shown are the mean ± S.E.
from two to eight experiments.
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Saturation Binding of EuK-c-Myc Antibodies to
1(c-Myc)
2
2,
1
2(c-Myc)
2, and
1
2
2(c-Myc)--
Saturation
binding experiments were carried out using control cells expressing
1
2
2 and cells expressing
receptors where either the
-,
-, or
-subunit was
epitope-tagged with c-Myc. Nonspecific binding of EuK c-Myc to control
cells expressing an untagged receptor was subtracted from total binding
at each concentration of antibody and was routinely 50% at 3 nM antibody. In all cases binding was saturable and had a
high affinity, and EuK-labeled c-Myc bound to more sites in the
1(c-Myc)
2
2 and
1
2(c-Myc)
2 compared with
1
2
2(c-Myc) as exemplified
in Fig. 3. To quantitate this, the
experiment was repeated on six separate occasions using 3 nM antibody, and the specific fluorescence counts are shown in Fig. 4A. The ratio of
fluorescence labeling to
1(c-Myc)
2
2,
1
2(c-Myc)
2, and
1
2
2(c-Myc) was
compared as shown in Fig. 4B. This clearly shows that there
are twice as many EuK c-Myc sites on receptors where the
- or
-subunit is tagged compared with receptors where the
-subunit is
tagged (ratio of
1(c-Myc)
2
2:
1
2(c-Myc)
2:
1
2
2(c-Myc) = 1.89 ± 0.12:1.94 ± 0.18:1).

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Fig. 3.
Saturation binding of EuK c-Myc to intact
cells expressing
1(c-Myc) 2 2,
1 2(c-Myc) 2,
and
1 2 2(c-Myc).
Data shown are specific fluorescence counts bound after subtraction of
nonspecific binding to cells expressing
1 2 2. Data shown are from
one experiment that was repeated three times with similar results. Data
were fitted using Excelfit, and the binding parameters obtained were:
Bmax = 515 fluorescence counts/fmol of receptors
and Kd = 6.2 nM for
1(c-Myc) 2 2 (open
circles); Bmax = 602 fluorescence
counts/fmol of receptors, Kd = 6.8 nM
for 1 2(c-Myc) 2
(closed circles); and Bmax = 169 fluorescence counts/fmol, Kd = 4.4 nM
for 1 2 2(c-Myc) (open
squares). For calculation of fluorescence counts/fmol of
receptors, a receptor was quantified using [3H]Ro15-1788
binding at 1.8 nM.
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Fig. 4.
Fluorescence signal observed at 620 and 665 nm from cells expressing
1(c-Myc) 2 2,
1 2(c-Myc) 2,
and
1 2 2(c-Myc).
Data shown are the mean ± S.E. of six independent experiments.
The specific fluorescence counts observed at 620 nm for
1(c-Myc) 2 2 were 315 ± 37/fmol and for
1 2(c-Myc) 2 were 286 ± 41/fmol. These were both significantly higher than that observed
with 1 2 2(c-Myc), 156 ± 21/fmol. The mean ± S.E. and the ratio for binding relative to
1 2 2(c-Myc) were calculated
for each experiment and are shown in panel B. The ratios of
binding for 1(c-Myc)
2 2: 1 2 2(c-Myc)
and
1 2(c-Myc) 2: 1 2 2(c-Myc)
were not significantly different from 2.0 (p < 0.05, Student's t test). Panel C, in the fluorescence
energy transfer experiments, where emission was measured at 665 nm,
there was no significant difference between the fluorescence counts
produced by 1(c-Myc) 2 2
(16.7 ± 1.5) and
1 2(c-Myc) 2 (17.8 ± 1.4), but both were significantly more than
1 2(c-Myc) (0.38 ± 2.4), which
was not significantly more than 0.
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A comparison of the Bmax values for
[3H]Ro15-1788 and EuK-c-Myc fluorescence can be used to
calculate the number of binding sites for the antibody/benzodiazepine
binding site. For
1(c-Myc)
2
2 there are
315 ± 37 fluorescence counts/fmol of Ro15-1788 binding sites
using 3 nM antibody and 1.8 nM
[3H]Ro15-1788 (Fig. 4). Using Clarke's equation
(occupancy = [ligand]/[ligand] + Ki) this
is equivalent to 610 ± 71 fluorescence counts/fmol of Ro15-1788
binding sites. Given that there are 418 fluorescence counts of EuK/fmol
of antibody (see "Experimental Procedures"), there are calculated
to be 1.46 ± 0.16 antibody binding
sites/[3H]Ro15-1788 binding site. The limitations and
multiple sources of error in this experiment (e.g.
aggregation of antibody, heterogeneity of antibody labeling, etc.)
would be more likely to lead to an underestimate of the number of
antibody binding sites therefore the observation that there is more
than one antibody binding site/[3H]Ro15-1788 binding site
supports the most likely stoichiometry of 2
-subunits/receptor:1
benzodiazepine site.
Fluorescence Energy Transfer Studies--
The affinity of the
EuK-derivatized c-Myc antibodies for the GABAA subunits was
4-6 nM as shown in Fig. 3. A concentration of 3 nM was selected for the fluorescence transfer studies
because this would less than half-maximally saturate the receptor.
Following incubation with 3 nM EuK-derivatized c-Myc mAb,
cells were washed and incubated with excess XL665-derivatized c-Myc
mAb, and fluorescence at 665 nm was measured in response to laser
excitation. As shown in Fig. 4C, a specific signal is
observed, indicating energy transfer when either the
1-
or the
2-subunit is epitope-tagged (16.7 ± 1.5 counts/fmol and 17.8 ± 1.4 counts/fmol) but not when the
2-subunit is epitope-tagged. Because energy transfer can
only take place when there are two c-Myc epitopes in close proximity, i.e. two subunits in the same receptor complex, this
confirms that the stoichiometry of the GABAA receptor under
study is
(
1)2(
2)2(
2)1. Furthermore, the lack of any observable signal with
1
2
2(c-Myc) could be
interpreted as evidence that receptors are not primarily expressed as
clusters on the cell surface. If this were the case, it may be possible
to observe energy transfer between
2-subunits on
separate receptors.
 |
DISCUSSION |
To date, the stoichiometry of the GABAA receptor has
been approached both directly and indirectly. Many laboratories have observed that a receptor can contain two different types of
-subunits (16-20). Conflicting evidence has been obtained on
whether two types of
-subunit can coexist in a single receptor
monomer with two studies proposing that two
-subunits can be present
(21, 22). There have been fewer studies on the presence of two
-subunits because the structural similarity of the
-subunits has
precluded the development of antibodies, which clearly distinguish
between them.
There have been four studies that directly analyzed the stoichiometry
of the receptor. The first two, chronologically, have taken an
electrophysiological approach. Backus et al. (23) favor the
composition 2×
, 1×
, and 2×
based on measuring the
outward current induced by point mutation of charged amino acids on
either side of the TM2 domain. Chang et al. (24) used a
similar method (mutation of a lysine in TM2) to increase the
sensitivity of the receptor to GABA in proportion to the number of
mutant subunits present. In contrast to Backus et al. (23),
they conclude that the stoichiometry is 2×
, 2×
, and 1×
,
as do Im et al. (25) from studies of tandem subunit
constructs. The most recent study used antibody labeling of chimeric
subunits to determine the ratio of subunits present from Western blots,
and they conclude that the structure is also 2×
, 2×
, and 1×
(11).
The approach taken here has used fluorescently derivatized mAbs to
quantify subunits relative to the benzodiazepine binding site on the
receptor and fluorescent resonance energy transfer to confirm that the
stoichiometry of the receptor is
(
1)2(
2)2
2. This method has several advantages over other previously described methods. 1) It uses intact cells; therefore only receptors that are
expressed on the surface (and are therefore presumed to be correctly
assembled) are considered (12, 26). 2) It involves minimal disruption
of receptor structure by requiring only epitope tagging, which does not
affect the expression or function of the receptor (12). 3) Only small
amounts of transiently transfected material are required.
Furthermore this approach has general applicability to other
multisubunit cell surface proteins and can be used particularly to
investigate the stoichiometry of subunits in GABAA
receptors composed of rarer subunits and in other ligand-gated ion channels.