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INTRODUCTION |
A very large number of membrane receptors modulate the activity of
intracellular effectors by activating heterotrimeric GTP-binding proteins (G-proteins).1 All
G-protein-coupled receptors characterized so far possess seven putative
transmembrane domains that define intracellular loops critical for the
recognition and activation of G-proteins. Sequence comparison revealed
the existence of at least three major families of G-protein-coupled
receptors. The members from different families share no sequence
similarity. Receptors homologous to rhodopsin (receptors for
catecholamines, acetylcholine, certain peptides, glycoproteins, etc.)
constitute the first family (family 1), which is to date the best
characterized one. Family 2 receptors are those homologous to the
vasoactive intestinal peptide and the glucagon receptors. Family 3 receptors comprise the metabotropic glutamate receptors (mGluRs) (1,
2), the Ca2+-sensing receptor (3), and a recently
discovered new family of putative pheromone receptors (4-6).
Family 3 G-protein-coupled receptors possess several unique features.
They all have a surprisingly large extracellular N terminus that shares
some sequence similarity with bacterial periplasmic amino acid-binding
proteins (PBPs) such as the leucine-binding protein (LBP) and the
leucine/isoleucine/valine-binding protein (LIVBP) (7). In agreement,
this portion of the receptor has been shown to play a critical role in
ligand recognition in mGluRs (7-10). Recently, the production of the
entire extracellular domain of mGluR1 in insect cells revealed that
this domain produced as a protein is able to fold correctly, is
soluble, and is sufficient to bind glutamate and its analogues in a
very similar manner as the wild-type receptor does (11). However,
further evidence that this domain shares a similar three-dimensional
structure with PBPs is still missing due to the lack of good
radioligands. The family 3 receptors also contain 20 conserved cysteine
residues; 17 are located in the N-terminal extracellular domain, 9 of
which are concentrated among the 100 residues that separate the
LBP-like domain and the first transmembrane domain. The functional
importance of this Cys-rich region has not yet been elucidated.
However, this region has been shown to be necessary for the LBP-like
domain of mGluR1 to bind glutamate (11). Other characteristic features of family 3 G-protein-coupled receptors are a highly conserved and
short third intracellular loop critical for G-protein activation (12)
and a variable second intracellular loop important for G-protein
coupling selectivity (13, 14).
The original structure of the binding domain of the family 3 receptors
led to a hypothesis for their mechanism of activation. PBPs are known
to be constituted of two lobes that close upon binding of the ligand,
like a Venus flytrap when touched by an insect (15, 16). It has
therefore been proposed that the two lobes of the LBP-like domain of
family 3 receptors also close upon binding of the agonist and that this
change in conformation is transduced to the transmembrane region to
activate the G-protein (7, 17). The agonist-binding domains of the
ionotropic glutamate receptor subunits are also homologous to PBPs (7,
18-21), and the closure of this domain upon agonist binding has also
been proposed to be responsible for receptor activation (22).
Recently, the cloning of the GABAB receptor revealed a
protein that is distantly related to family 3 receptors. Indeed the GABAB receptor does not share all of the characteristic
features of family 3 receptors described above (23). Like other family 3 receptors, the GABAB receptor possesses a large
extracellular domain that shares some limited but significant
similarity with PBPs such as LBP. However, the intracellular loops of
the GABAB receptor are not as well conserved as in the
other family 3 receptors. Moreover, the cysteines in the LBP-like
domain, highly conserved in the other family 3 receptors, are not
present in the GABAB receptor, and no Cys-rich region is
found in this receptor. Because high affinity and specific radioligands
are available for the GABAB receptor and because this
receptor is related to the family 3 G-protein-coupled receptors, it
appears to be a good model for a further analysis of the possible
structure of the extracellular domain of this new receptor family. This
would help identify the possible role of this domain in ligand
recognition and receptor activation.
Here, we report the generation of a model for the
GABAB-binding domain and its assessment by site-directed
mutagenesis. This study shows that the extracellular domain of the
GABAB receptor likely folds into two lobes separated by a
hinge region like the PBPs and identifies specific residues within this
domain important for ligand recognition.
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EXPERIMENTAL PROCEDURES |
Materials--
-Amino-n-butyric acid (GABA) was
obtained from Sigma (L'Isle d'Abeau, France).
125I-CGP64213 was synthesized from ethyl
(1,1-diethoxyethyl)phosphinate as described (24) and labeled to a
specific radioactivity of >2000 Ci/mmol (ANAWA AG, Wangen,
Switzerland). All other ligands were synthesized in-house (24, 25).
Dithiothreitol (DTT) was from Roche Molecular Biochemicals (Mannheim,
Germany). Serum, culture media, and other solutions used for cell
culture were from Life Technologies, Inc. (Cergy Pontoise, France). The
polyclonal anti-GABAB-R1 antibody was raised against the
C-terminal intracellular part of the receptor as described previously
(26). A secondary fluorescein-labeled anti-rabbit antibody (Sigma) was
used for immunostaining. The ECL chemiluminescence system (Amersham
Pharmacia Biotech ) was used to detect the secondary antibody on
Western blots.
Sequence Alignment--
A multiple alignment of the
metabotropic glutamate receptor (rat mGluR1-8 plus the
Drosophila receptor DmGluRA) and
Ca2+-sensing receptor LBP-like domains according to O'Hara
et al. (7) and of the bacterial periplasmic proteins that
bind leucine/isoleucine/valine (LIVBP) and leucine (LBP) was generated
using the ClustalW Version 1.60 program (27). A gap penalty of 30, an
extension gap penalty of 0.05, and the Blosum30 matrix were used. The
alignment was then manually modified in order not to introduce gaps
into the sequence that aligned with the known secondary structure
elements of LIVBP and LBP. On top of the multiple alignment, part of
the extracellular domain of GABAB-R1a (residues 109-558)
was then aligned using the profile alignment command of the ClustalW
Version 1.60 program and a gap penalty ranging from 2 to 50. These
alignments indicated that the LBP-like domain of GABAB-R1a
maps from residues 164 to 550. This later segment of the
GABAB-R1a sequence was thereafter used to perform
additional alignments. These were further analyzed and manually
modified using the hydrophobic cluster analysis (28). The resulting
alignments of the extracellular GABAB-binding site, LIVBP,
and LBP were then used to generate three-dimensional models using the
program Modeler (see below). The coordinates of these models were then
subjected to the Verify3D algorithm (29) using the Verify3D Structure
Evaluation Server2 to
identify regions of improper folding. After further manual modifications, the alignment giving rise to models with better Verify3D
scores was selected.
Molecular Modeling--
The three-dimensional model of the
GABAB-binding domain was constructed by homology using the
coordinates of both LIVBP and LBP from Escherichia coli
(Protein Data Bank accession numbers 2LIV and 2LBP, respectively)
obtained by x-ray crystallography. The sequence alignment used was
obtained as described above. Several models were generated using the
program Modeler (30) in the Insight-II environment (Molecular
Simulation Inc., San Diego, CA) on a Silicon Graphics R10000 O2
workstation. The hydrophobic cluster analysis suggested that part of
the insertion found just after
-helix 9 (see Fig. 1) also likely
folds into an
-helix. Accordingly, new models were generated in
which parts of the insertion (residues 398-406 and 412-418) were
folded into an
-helix. Accordingly, the side chains of Cys-375 and
Cys-409 were in close proximity, making them likely to form a disulfide
bond. This was further imposed during the modeling procedure. These
modifications improved the scores obtained in this area of the sequence
with the Verify3D algorithm (29). The model giving the best scores was
selected and subjected to energy minimization using the program
Discover Version 2.9.7 (Molecular Simulation Inc.) and the CVFF force
field. A steepest descent followed by a conjugate gradient method was applied (without taking into account the electrostatic term) until the
maximum energy derivative was <1.0
kcal·Å
1·mol
1.
Site-directed Mutagenesis and Expression in HEK-293
Cells--
Single amino acid replacement was carried out by the Quick
Change method (Stratagene, La Jolla, CA) according to the
manufacturer's instructions using pBSB5 as a template. This vector was
constructed by subcloning the coding sequence of GABAB-R1a
into the EcoRI and NcoI restriction sites of
pBluescript SK (Stratagene). For each mutagenesis, two complementary
30-mer oligonucleotides (sense and antisense) were designed to contain
the desired mutation in their middle. To allow a rapid screening of the
mutated clones, the primers carried an additional silent mutation
introducing (or removing) a restriction site. The presence of each
mutation of interest and the absence of undesired ones were confirmed
by DNA sequencing. Subsequently, a short fragment surrounding the mutation has been subcloned in place of the corresponding wild-type fragment of pRKBR1a (an expression vector constructed by inserting the
open reading frame of GABAB-R1a downstream of the
cytomegalovirus promoter of pRK5).
Wild-type and mutated expression constructs were transfected into
HEK-293 cells by electroporation as described previously (31).
Electroporation was carried out in a total volume of 300 µl with 10 µg of carrier DNA, 2 µg of plasmid DNA containing the wild-type or
mutated GABAB-R1a coding sequences, and 10 × 106 cells. The cells were cultured in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
calf serum and antibiotics.
Ligand Binding Assay--
Ligand competition experiments were
performed on membranes of HEK-293 cells prepared as followed. 24 h
after transfection, the cells were transferred into serum-free
Dulbecco's modified Eagle's medium; 48 h after transfection, the
cells were washed and homogenized in Tris/Krebs buffer (20 mM Tris-Cl (pH 7.4), 118 mM NaCl, 5.6 mM glucose, 1.2 mM
KH2PO4, 1.2 mM MgSO4,
4.7 mM KCl, and 1.8 mM CaCl2) and
centrifuged for 20 min at 40,000 × g. The pellet was
resuspended in Tris/Krebs buffer and stored at
80 °C. For ligand
competition assays, thawed membranes (10 µg of protein) were
incubated with 0.1 nM 125I-CGP64213 in the
presence of unlabeled ligands at the indicated concentrations.
Nonspecific binding was determined using 10 mM GABA or 100 nM CGP54626A. Incubation was terminated by filtration through Whatman GF/C glass-fiber filters. The curves were fitted with
Kaleidagraph software using the following equation: y = ((ymax
ymin)/(1 + (x/IC50)nH)) + ymin, where IC50 is the cold drug
concentration necessary to displace half of the specifically bound
125I-CGP64213 and nH is the Hill number.
Western Blot and Immunofluorescence Experiments--
For Western
blotting, HEK-293 cell membranes prepared as described above were
solubilized in Laemmli sample buffer (2% (w/v) SDS, 50 mM
Tris-HCl (pH 6.8), 50 mM DTT, 0.1% (v/v) bromphenol blue,
and 10% glycerol), and after 5 min of warming at 37 °C, 1 µg of
protein was resolved by SDS-polyacrylamide gel electrophoresis (10%
acrylamide) and transferred by electroblotting onto a Hybond C extra
membrane (Amersham Life Sciences). The primary polyclonal antibody used
for the immunodetection of the GABAB-R1a protein was
described previously (26). Protein concentrations were measured with
the Bradford method using the Bio-Rad protein assay reagent. The
immunofluorescence experiments were performed 18 h after
transfection on HEK-293 cells as described previously (32).
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RESULTS AND DISCUSSION |
Identification and Molecular Modeling of the LBP-like Domain of the
GABAB Receptor--
Sequence comparison between the
extracellular domain of the GABAB receptor, the
metabotropic glutamate receptors, and PBPs reveals that the sequence
between residues 167 and 550 of GABAB-R1a shares sequence
similarity with the LBP-like domain of mGluRs (18.9% with mGluR1 and
15.8% with the Ca2+-sensing receptor) and LBP and LIVBP
(38.1 and 37.1%, respectively) (Fig.
1a). GABAB-R1a and
GABAB-R1b are two splice variants that differ in their
N-terminal sequence. N-terminal residues 1-163 of
GABAB-R1a are replaced by 47 different residues in
GABAB-R1b (Fig. 1b) (23). Interestingly, the
similarity to the LBP and LIVBP proteins starts just after the splice
junction site, suggesting that the LBP-like domain of GABAB
receptors starts at the beginning of the first exon common to both
GABAB-R1a and GABAB-R1b. In agreement with this
proposal, the specific sequence of GABAB-R1a folds in a
tandem pair of complement protein modules (also known as sushi repeats)
(33-35), the first corresponding to Thr-25-Arg-97 and the second to
Ile-84-Asn-159. The second sushi-like domain of GABAB-R1a
ends almost exactly before the splice site. This reveals that
GABAB-R1a and GABAB-R1b share an identical
LBP-like domain (Fig. 1b). Accordingly and in agreement with
the hypothesis that the LBP-like domain constitutes the ligand
recognition domain of family 3 receptors (7-11), the two
GABAB receptor variants have identical pharmacological
profiles (23). Sequence comparison reveals that the large insertions
found in the LBP-like domain of mGluRs (I1, I2,
and I3 in Fig. 1a) are not found in the
GABAB receptor, further indicating that the
GABAB receptor is most distantly related to the other
family 3 receptors. Our sequence alignment also confirms that most of
the extracellular domain of GABAB-R1b is homologous to LBP
(Fig. 1b). A short sequence of only 39 residues links this
domain to the first transmembrane domain.

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Fig. 1.
The extracellular domain of GABAB
receptors shares sequence homology with PBPs such as LBP and LIVBP.
a, amino acid sequence alignment of the LBP-like domain of
mGluR1, LBP, and LIVBP from E. coli and the extracellular
domain of GABAB-R1a. The large insertions found in the
LBP-like domain of mGluR1 have been removed, and their positions are
indicated by I1 (10 residues), I2 (25 residues),
and I3 (33 residues). The -strands (thin
lines) and -helices (thick lines) identified in LBP
and LIVBP crystal structures (36, 37) are indicated on top of the
alignment. The position of the linkers that interconnect the two lobes
of LBP and LIVBP are indicated by L1, L2, and
L3 on top of the alignment. Residues that constitute the
leucine-binding pocket in LIVBP are indicated by open and
closed circles on top of the alignment. Closed
circles indicate residues that form hydrogen bonds with the
-carboxylic (Ser-79) and -amino (Thr-102) groups of leucine in
LIVBP (36). Closed circles also indicate the residues that
affect agonist potency in mGluR1 (Ser-165 and Thr-188) (7). Cys
residues that are involved in disulfide bonds in LIVBP and LBP and that
are possibly involved in disulfide bonds in the GABAB
receptor are linked. The positions of the N-glycosylation
sites in the GABAB receptor are indicated by N
under the alignment. Residues that have been subjected to site-directed
mutagenesis in the GABAB receptor are indicated: Open
square, Cys-187, does not affect binding; closed
squares, Cys residues the mutation of which suppresses binding;
open triangles, no effect on binding; closed
triangles, Ser-246 and Tyr-470, suppress binding; plus
signs, Ser-247 and Gln-312, increase agonist affinity;
asterisks, Ser-269 and Ser-270, affect the affinity of some
ligands. b, schematic representation of the family 3 receptors. S, signal peptide; LBP-like, LBP-like
domain; Cys-rich, cysteine-rich domain; Sushis,
short consensus repeats or sushi domains; 7TMD,
seven-transmembrane domain; C-term, intracellular
carboxyl-terminal domain. CaR, Ca2+-sensing
receptor; PheromoneR, pheromone receptor.
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A three-dimensional model was generated using the known
three-dimensional structure of the open state of LIVBP and LBP (Fig. 2). This model was assessed using the
Verify 3D algorithm according to Luthy et al. (29). As shown
in Fig. 3, the three/one-dimensional scores of our model are always positive and are similar to those obtained with the template structures of LBP and LIVBP. The scores obtained are in the range of scores for highly refined correct X-ray
structure determinations. This indicates that this domain of the
GABAB receptor can fold like the LBP protein. As a further validation of the model, all Asn residues that are part of a consensus glycosylation site (Fig. 1a) were found to be at the surface
of the protein (brown residues in Fig. 2b). This
model reveals that the LBP-like domain of the GABAB
receptor is composed of two lobes linked by three short linkers (Fig.
2a). Both lobes comprise an alternation of
-helices and
-sheets, one lobe having three additional helices. The latter will
be referred to as lobe I, and the other as lobe II (Fig. 2).

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Fig. 2.
Three-dimensional model of the LBP-like
domain of the GABAB receptor. a, ribbon
representation of the LBP-like domain of the GABAB
receptor. Residues that have been mutated and found to play a role in
CGP64213 binding are shown. The linkers that interconnect the two lobes
(L1, L2, and L3) are indicated.
b, Corey-Pauling-Koltun representation of the LBP-like
domain of the GABAB receptor. In red is Ser-246,
the mutation of which suppresses binding of 125I-CGP64213;
in blue are Ser-247 and Gln-312, the mutation of which
results in an increase in affinity of agonists; in violet
are Ser-269 and Ser-270, the mutation of which differently affects
binding affinities of various ligands; in yellow are the
four Cys residues likely forming disulfide bonds; and in
brown are the Asn residues likely to be glycosylated.
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Fig. 3.
Plots obtained using the Verify3D program
with a window of 21 residues using the coordinates of the LBP and LIVBP
crystal structures (Protein Data Bank accession numbers 2LBP and 2LIV,
respectively) (top) and the coordinates of the
three-dimensional model of the LBP-like domain of the GABAB
receptor (bottom). The vertical gray
bars in the plot for LBP and LIVBP correspond to the insertions
found in the GABAB receptor domain. The x axis
numbering corresponds to the amino acid numbering of
GABAB-R1a. The y axis gives the
average three/one-dimensional scores for residues in a 21-residue
sliding window.
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Importance of Cysteine Residues for 125I-CGP64213
Binding--
Among the five cysteine residues found in the
extracellular domain, the model predicts that Cys-219 and Cys-245 as
well as Cys-375 and Cys-409 are in close proximity and likely form
disulfide bonds (Fig. 2a) (the residue numbers correspond to
those of the GABAB-R1a sequence, with the first Met being
1; this numbering will be used throughout this work). Cys-187 is buried
inside lobe I and cannot be involved in a disulfide bond (Fig.
2a). Cys-219 and Cys-245 correspond to Cys-53 and Cys-78 of
LIVBP and LBP, which are disulfide-bonded (36, 37). This putative
disulfide bridge found in the GABAB receptor is not found
in the other family 3 receptors since only one of the Cys
residues is conserved (see Fig. 1a for the mGluR1 sequence).
To examine if these Cys residues are important for the correct folding
of the protein and/or for the binding of GABAB receptor ligands, they were changed into either Ala or Ser by in
vitro mutagenesis. The mutation of Cys-187 did not prevent the
binding of the GABAB receptor antagonist
125I-CGP64213 (Table I), and
displacement curves indicated that the affinities for the agonists GABA
and APPA and for the antagonists CGP64213 and CGP54626A were not
affected by these mutations (Table I and data not shown). Both mutants
C187A and C187S were expressed and had the expected molecular mass,
although C187S expression levels were lower than those of the wild-type
receptor (Fig. 4). In contrast, mutation
of any of the other four Cys residues into either Ala or Ser or the
double mutation of Cys-219 and Cys-245 or of Cys-375 and Cys-409
totally prevented the binding of 125I-CGP64213 to the
receptor (Table I). In any case, the mutant proteins with a correct
molecular mass could be detected on Western blots, although some (C245S
and C219A/C245A) were expressed at very low levels (Fig. 4). These data
indicate that the loss of binding is not due to a loss of expression or
a rapid degradation of the protein.
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Table I
Effect of the C187A and C187S mutations on the binding affinities of
GABA and CGP54626A
Binding experiments were performed using 0.1 nM
125I-CGP64213 as described under "Experimental
Procedures." Displacement curves performed with 11 different
concentrations of GABA or CGP54626A were fitted as described under
"Experimental Procedures." Concentrations giving rise to 50%
inhibition of specific binding (IC50) and Hill coefficients
(nH) were determined. Values are means ± S.E.
of at least three experiments performed in triplicates.
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Fig. 4.
Expression of the Cys mutants of
GABAB-R1a. Membranes (1 µg of total protein)
prepared from mock-transfected HEK-293 cells or cells expressing
wild-type (Wt) GABAB-R1a or the indicated
mutated receptors were subjected to SDS-polyacrylamide gel
electrophoresis and blotted as described under "Experimental
Procedures." GABAB-R1a proteins were detected using a
polyclonal antibody directed against the carboxyl-terminal region of
the protein. The specific binding of 125I-CGP64213 was
determined on the same membranes and is indicated at the top.
0, no specific binding was detected.
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These data are in agreement with our model predicting that these
residues are involved in two disulfide bonds. We were therefore expecting that the binding of 125I-CGP64213 would be
sensitive to reducing conditions. However, as shown in Fig.
5, membrane treatment with 10-100
mM DTT did not suppress 125I-CGP64213 binding.
This indicates that, if there are disulfide bonds, they are not
required for proper binding after the protein has been synthesized and
correctly folded. To explain the lack of binding observed with the Cys
mutants, one may propose that these disulfide bonds are required for
the correct folding of the protein during synthesis. After the protein
is fully matured, these covalent links may only increase the stability
of this domain of the receptor. We therefore studied the
thermostability of the binding site in the presence or absence of 10 mM DTT (Fig. 5). Exposure of the wild-type receptor for a
period of 30 min at temperatures ranging from 25 to 65 °C revealed
that 50% of the 125I-CGP64213 binding disappeared at
50 °C. This temperature was found to be 5.2 ± 0.1 °C
(n = 3) lower after DTT treatment (Fig. 5). These
results are consistent with the hypothesis that disulfide bonds are
involved in the stability of the binding domain of the GABAB receptor. However, we cannot exclude the possibility
that the DTT effect results from its action on another protein
stabilizing the GABAB receptor-binding site.

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Fig. 5.
Effect of DTT treatment on the
thermostability of the CGP64213-binding site. Membranes (5 µg)
prepared from cells expressing wild-type GABAB-R1a were
incubated for 30 min with (open squares) or without
(closed circles) 10 mM DTT at the temperatures
indicated on the graph. Then membranes were pelleted and washed twice
with Tris/Krebs buffer. The binding experiment was then performed as
described under "Experimental Procedures." The values correspond to
the specific binding and are expressed as percent of that obtained with
membrane preincubated at 25 °C. Results are means ± S.E. of
triplicate determinations from a representative experiment.
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Selection and Mutagenesis of Additional Residues Possibly Involved
in 125I-CGP64213 Binding--
The amino acid residues of
LIVBP (open state) involved in the binding of leucine have been
identified by x-ray crystallography and were found in lobe I (36). The
side chain of Ser-79 and Thr-102 have been shown to form hydrogen bonds
with the
-amino and
-carboxylic groups of leucine. Base on this
information, it has been shown that the homologous residues in the
mGluR1-binding domain, Ser-165 and Thr-188, are involved in the binding
of glutamate. Mutation into alanine decreases the glutamate potency by
100- and 10,000-fold, respectively (7). In LIVBP, the side chain of
leucine interacts in a hydrophobic pocket formed by Tyr-18, Ala-100,
Ala-101, and Phe-276 (36). To try to identify the amino acid residues
possibly involved in the binding of GABAB receptor ligands,
a set of point mutations around residues homologous to those that
constitute the leucine-binding pocket in LIVBP have been generated
(Figs. 1a and Fig. 2). Some residues located on lobe II and
facing the putative binding pocket were also mutated (residues
corresponding to the
6-
6 loop; see Figs. 1a and
2b).
Among these 20 different mutations, only six were found to change the
binding properties of the GABAB receptor (Fig. 2 and Table
II). These include S246A, S247A, S269A,
S270A, Q312A, and Y470A. All these mutated receptors were detected on
Western blots as proteins with the correct molecular mass (Fig.
6) and had the same subcellular
distribution as the wild-type receptor (Fig. 7), indicating that the changes in their
binding properties were not due to a loss of correct expression or
trafficking.
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Table II
Effect of various mutations of GABAB-R1a on the binding
affinities of GABA and CGP54626A
Binding experiments were performed using 0.1 nM
125I-CGP64213 as described under "Experimental Procedures."
Displacement curves performed with 11 different concentrations of GABA
or CGP54626A were fitted as described under "Experimental
Procedures." Concentrations giving rise to 50% inhibition of
specific binding (IC50) and Hill coefficients
(nH) were determined. Values are means ± S.E.
of at least three experiments performed in triplicates. wt, mutants in
which the IC50 values of GABA and CGP54626A were similar to
those of the wild-type receptor; , mutants in which the affinity of
some ligands was decreased (see Fig. 8); +, mutants in which the
affinity of agonists was increased.
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Fig. 6.
Expression of some mutants of
GABAB-R1a. Membranes (1 µg) prepared from
mock-transfected HEK-293 cells or cells expressing wild-type
(Wt) GABAB-R1a or the S246A, S247A, S270A,
S269A, Q312A, or Y470A mutated receptor where subjected to
SDS-polyacrylamide gel electrophoresis as described under
"Experimental Procedures." The receptor proteins were detected
using a polyclonal antibody directed against the carboxyl-terminal
region of the protein. The specific binding of
125I-CGP64213 was determined on the same membranes and is
indicated at the top. 0, no specific binding was
detected.
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Fig. 7.
Fluorescence immunostaining of wild-type
GABAB-R1a and mutated receptors in HEK-293
cells. Cells expressing the wild-type receptor (wt;
a), mock-transfected cells (b), and cells
expressing the S246A (c) or Y470A (d) mutated
receptor were immunostained with the same polyclonal antibody used for
Western blotting, followed by a fluorescein-coupled goat anti-rabbit
secondary antibody.
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Mutations That Suppress Binding--
Two of these mutations, S246A
and Y470A, resulted in a total loss of the antagonist
125I-CGP64213 binding, even when a 10-fold higher
concentration of the radioligand (1 nM) was used (data not
shown). Ser-246 aligns with Ser-79 of LIVBP and Ser-165 of mGluR1 (Fig.
1), which have been shown and proposed to form a hydrogen bond with the
-carboxylic group of leucine and glutamate in LIVBP and mGluR1,
respectively (7, 36). It is therefore possible that Ser-246 is directly involved in the binding of agonists and antagonists. Tyr-470 is conserved in all family 3 receptors, LIVBP, and LBP (Fig.
1a). As the side chain of Tyr-470 is located inside lobe I
(Fig. 2a), it is unlikely directly interacting with the
ligand. Instead, the removal of the phenyl ring may simply affect the
conformation of lobe I. Assuming that this lobe contains the binding
site, this would affect ligand binding. In agreement with this
proposal, mutation of Tyr-470 into phenylalanine restored binding and
affinity for 125I-CGP64213 and GABA, although binding was
difficult to detect due to a very low expression of the mutant protein
(data not shown).
Mutations That Differently Affect Binding Affinities of Various
Ligands--
The mutations S269A and S270A affected the affinities of
some but not all ligands (residues shown in violet in Fig.
2b). In mutant S269A, the affinity of the antagonist
CGP54626A was decreased by a factor of 50, whereas that of the
antagonists CGP64213 and CGP56999A was affected by a factor of only 5 (Fig. 8 and Table II). Similarly, the
affinities of the three agonists GABA, APPA, and CGP47656 were
decreased by 11-, 30-, and 10-fold, respectively, whereas the affinity
of baclofen possessing an additional chlorophenyl group was not
affected (Fig. 8 and Table II). In mutant S270A, the affinities of the
agonists GABA, APPA, and baclofen were decreased by a factor of >10,
whereas those of the antagonists CGP54626A, CGP64213, and CGP56999A
were not affected significantly (Fig. 8 and Table II). Due to the
relatively small changes in affinity observed, it is unlikely that
these two residues are directly involved in an interaction with the
ligands. Ser-269 aligns with Thr-102 of LIVBP and Thr-188 of mGluR1
(Fig. 1a). These residues have been shown and proposed to
form a hydrogen bond with the
-amino group of leucine and glutamate
in LIVBP and mGluR1, respectively (7, 36). Accordingly, mutation of
Thr-188 into Ala in mGluR1 resulted in a decrease in the quisqualate
potency by a factor of 10,000. GABA lacks the typical amino acid moiety
found on the
-carbon of both leucine and glutamate. It is therefore
not surprising that Ser-269 of the GABAB receptor does not
play a role similar to that of Thr-102 and Thr-188 of LIVBP and mGluR1,
respectively.

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Fig. 8.
Displacement curves for the S269A, S270A,
S247A, and Q312A mutants with the agonists GABA, APPA, and baclofen and
the antagonists CGP54626A, CGP64213, and CGP56999A. All
experiments were carried out with 0.1 nM
125I-CGP64213 displaced by unlabeled ligands at the
concentrations indicated on the graph. Closed circles, GABA;
open squares, APPA; closed triangles, baclofen;
open circles, CGP54626A; closed squares,
CGP64213; open triangles, CGP56999A. The dashed
lines correspond to the positions of the IC50 values
of each drug (represented by the appropriate symbol at the bottom of
each graph) obtained with the wild-type receptor. Each curve is the
mean of three independent experiments performed in triplicates.
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Mutations That Increase Agonist Affinities--
Finally, both
mutations S247A (lobe I) and Q312A (lobe II) (blue residues
in Fig. 2b) resulted in an increase in agonist affinity (GABA, APPA, and baclofen) by a factor of >3 and in a decrease in
antagonist affinity (CGP54626A, CGP64213, and CGP56999A) by a factor of
2 (Fig. 8 and Table II). The changes in affinity were too small to
assume that Ser-247 and Gln-312 directly interact with these ligands.
How can one explain the opposite effect of these two mutations on the
binding affinities of agonists and antagonists? One possible
explanation, as detailed below, is that these mutations affect the
closure of the LBP-like domain of the GABAB receptor. As
mentioned in the Introduction, PBPs adopt two conformational states, an
open and a closed state. Binding of the ligand is known to favor the
closed state according to Model I (38-42),
where Rc is the closed state of the protein, Ro is
the open state, K2 is the equilibrium constant
between the closed and open states (K2 = [Rc]/[Ro]), K1 is the
binding affinity of the ligand (L) on the open state, and
is the
change in the equilibrium constant between Rc and Ro
when the ligand is bound on the protein. According to this model, the
affinity constant of a ligand (Kd) is as follows:
Kd = ([Ro] + [Rc])·[L]/([RoL] + [RcL]), and is
therefore equal to Kd = K1(1 + K2)/(1 +
K2).
According to the similarity between the GABAB
receptor-binding domain and PBPs, the same model can be proposed for
the GABAB receptor. If we consider that the closure of the
LBP-like domain is a necessary step for receptor activation, the factor
must be higher than 1 for agonists (the agonist stabilizes the
closed state). In contrast,
may be lower or equal to 1 in the case of antagonists (they do not favor or even prevent the formation of the
closed state). According to the above equation, an increase in
K2 due to a mutation in the receptor will result
in a decrease in the Kd for an agonist and an
increase in Kd for an antagonist, as observed
for the S247A and Q312A mutations.
As shown in the three-dimensional model of the open state of the
GABAB-binding site (Fig. 2, a and b),
Ser-247 and Gln-312 are located in such a position that they can be in
close proximity to the other lobe in the closed state (43).
Accordingly, mutation of these residues may affect
K2 and therefore have opposite effects on the
affinities of agonists and antagonists. In agreement with this
proposal, amino acids at comparable positions in PBPs such as the
histidine-binding protein have been shown to affect the equilibrium
constant between the closed and open states (40, 41, 44). It is
interesting to note here that Ser-247 aligns with Ser-73 in LBP and
Ser-166 in mGluR1. This latter residue in mGluR1 is responsible for the
calcium-induced activation of mGluR1 (38).
Conclusion--
Taken together, our study shows that the structure
of the GABAB receptor-binding site is likely to be similar
to that of PBPs. Our three-dimensional model predicts that four Cys
residues are important for the structural stability of this domain in
the GABAB receptor and is confirmed by mutations of these
residues and by the decrease in the thermostability of the binding site
resulting from DTT treatment. In agreement with the three-dimensional
model, 14 different mutations do not affect the binding properties of the receptor, and one residue (Ser-246) that is likely to be directly involved in the binding of GABAB receptor ligands has been
identified. These data support a Venus flytrap model for the
GABAB receptor activation, as previously proposed for the
other family 3 receptors.