Locking the Dimeric GABAB G-Protein-Coupled Receptor in Its Active State

Abstract

G-protein-coupled receptors (GPCRs) play a major role in cell-cell communication in the CNS. These proteins oscillate between various inactive and active conformations, the latter being stabilized by agonists. Although mutations can lead to constitutive activity, most of these destabilize inactive conformations, and none lock the receptor in an active state. Moreover, GPCRs are known to form dimers, but the role of each protomer in the activation process remains unclear. Here, we show that the heterodimeric GPCR for the main inhibitory neurotransmitter, the GABA(B) receptor, can be locked in its active state by introducing two cysteines expected to form a disulphide bridge to maintain the binding domain of the GABA(B1) subunit in a closed form. This constitutively active receptor cannot be inhibited by antagonists, but its normal functioning, activation by agonists, and inhibition by antagonists can be restored after reduction with dithiothreitol. These data show that the closed state of the binding domain of GABA(B1) is sufficient to turn ON this heterodimeric receptor and illustrate for the first time that a GPCR can be locked in an active conformation.

Full text

Cellular/Molecular
Locking the Dimeric GABA
B
G-Protein-Coupled Receptor in
Its Active State
Julie Kniazeff,
1
Pierre-Philippe Saintot,
1
Cyril Goudet,
1
Jianfeng Liu,
1
Annie Charnet,
2
Gilles Guillon,
2
and
Jean-Philippe Pin
1
1
Laboratory for Functional Genomic, Department of Molecular Pharmacology, Centre National de la Recherche Scientifique Unite´ Propre de Recherche-
2580, and
2
Institut National de la Sante´ et de la Recherche Me´dicale U469, 34094 Montpellier Cedex 5, France
G-protein-coupled receptors (GPCRs) play a major role in cell– cell communication in the CNS. These proteins oscillate between various
inactive and active conformations, the latter being stabilized by agonists. Although mutations can lead to constitutive activity, most of
these destabilize inactive conformations, and none lock the receptor in an active state. Moreover, GPCRs are known to form dimers, but
the role of each protomer in the activation process remains unclear. Here, we show that the heterodimeric GPCR for the main inhibitory
neurotransmitter, the GABA
B
receptor, can be locked in its active state by introducing two cysteines expected to form a disulphide bridge
to maintain the binding domain of the GABA
B1
subunit in a closed form. This constitutively active receptor cannot be inhibited by
antagonists, but its normal functioning, activation by agonists, and inhibition by antagonists can be restored after reduction with
dithiothreitol. These data show that the closed state of the binding domain of GABA
B1
is sufficient to turn ON this heterodimeric receptor
and illustrate for the first time that a GPCR can be locked in an active conformation.
Key words: receptor; baclofen; GABA; metabotropic glutamate receptor; glutamate; activation mechanism
Introduction
G-protein-coupled receptors (GPCRs) play a major role in cell–
cell communication, especially in the brain, where they are in-
volved in the tuning of fast synaptic transmission. Several classes
of GPCRs can be defined based on sequence similarity (Kola-
kowski, 1994; Bockaert and Pin, 1999; Fredriksson et al., 2003).
GPCRs for the two main neurotransmitters, GABA and gluta-
mate [the GABA
B
and metabotropic glutamate receptors
(mGluRs)] are part of the class III GPCRs, together with those
activated by Ca
2⫹
, sweet molecules, and some pheromones (Pin
et al., 2003). These receptors form dimers, but the GABA
B
recep-
tor is an obligatory heterodimer constituted of the homologous
GABA
B1
and GABA
B2
subunits (Jones et al., 1998; Kaupmann et
al., 1998; White et al., 1998). GABA
B1
is responsible for GABA
recognition (Galvez et al., 2000a; Kniazeff et al., 2002), whereas
GABA
B2
is necessary for the correct trafficking of GABA
B1
to the
cell surface (Margeta-Mitrovic et al., 2000; Pagano et al., 2001)
and is involved in G-protein activation (Galvez et al., 2001;
Margeta-Mitrovic et al., 2001b; Robbins et al., 2001; Duthey et al.,
2002).
The ligand-binding site of class III GPCRs is located in a large
extracellular domain (Takahashi et al., 1993) homologous to
some periplasmic-binding proteins (O’Hara et al., 1993) and to
the binding domain of other receptors such as ionotropic gluta-
mate, atrial natriuteric peptide (ANP), and some tyrosine kinase
receptors (Felder et al., 1999; Vicogne et al., 2003). X-ray struc-
tures have been solved for the extracellular domains of mGluR
type 1 (mGlu1) (Kunishima et al., 2000; Tsuchiya et al., 2002) and
ANP receptor (ANPR) types A and C (He et al., 2001; van den
Akker, 2001) produced as soluble proteins. These domains are
bilobate proteins often called Venus flytrap modules (VFTMs)
and share similar sequence homology with the extracellular do-
main of the GABA
B
subunits (Fig. 1a). The mGlu1 VFTM was
observed either in an open or closed conformation, whereas that
of the ANPR is only found in a closed conformation.
Both mGlu1 and ANP VFTMs homodimerize at the level of
their lobe I, leaving the two lobe II (connected to the membrane
domain) far apart in the absence of agonists (Fig. 1b,c). In the
presence of agonists, a change in conformation brings together
lobe II, possibly forcing the membrane domains to interact dif-
ferently (Fig. 1b,c). In the case of mGlu1, this results from: (1)
closure of at least one VFTM; and (2) a change in the relative
orientation of the VFTMs (Fig. 1b). In the case of the ANPR, this
results from the binding of ANP between the two VFTMs (Fig.
1c). Whether the dimer of GABA
B
VFTMs functions like that of
mGlu or ANPRs remains unknown.
Here, we show that the introduction of two cysteines expected
to lock the GABA
B1
VFTM in a closed state by a disulphide bridge
is sufficient to lock the entire receptor in an almost fully active
Received July 1, 2003; revised Nov. 10, 2003; accepted Nov. 10, 2003.
This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS), Action Con-
certe´e Incitative “Mole´cules etCibles The´rapeutiques” from Institut Nationalde la Sante´ et de laRecherche Me´dicale,
and the French government (J.P.P.). J.L. and A.C. were supported by Groupement d’Intéreˆt Public Hoechst Marion
Roussel/Aventis and CisBio International, respectively. J.K. and C.G. were supported by CNRS and Fondation pour la
Recherche Me´dicale fellowships, respectively. We thank Drs. T. Galvez, G. Labesse, J. Neyton, M. L. Parmentier, L.
Pre´zeau, and P. Paoletti for constructive discussions and critical reading of this manuscript. We also thank Drs. W.
Froestl and K. Kaupmann (Novartis, Basel, Switzerland) for the supply of GABA
B
ligands and Dr. P. Rondard for the
construction of ⌬GB1 and ⌬GB2.
Correspondence should be addressed to Dr. Jean-Philippe Pin, Laboratory for Functional Genomic, Centre Na-
tional de la Recherche Scientifique Unite´ Propre de Recherche-2580, Department of Molecular Pharmacology, 141
rue de la Cardonille, F-34094 Montpellier Cedex 5, France. E-mail: jppin@ccipe.cnrs.fr.
DOI:10.1523/JNEUROSCI.3141-03.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/240370-08$15.00/0
370 •The Journal of Neuroscience, January 14, 2004 •24(2):370 –377

state. This reveals a similar activation mechanism for mGlu and
GABA
B
receptors and represents the first example of a GPCR that
has been locked in its active state. As such, this study brings much
information on the way the GPCRs for the main neurotransmit-
ters, glutamate and GABA, are activated.
Materials and Methods
Materials. GABA, baclofen, and CGP64213 were gifts from Drs.
W. Froestl and K. Kaupman (Novartis Pharma, Basel, Switzerland).
[
125
I]CGP64213 was purchase from Anawa (Zurich, Switzerland). FBS,
culture media, and other solutions used for cell culture were from
Invitrogen-Life Technologies, Inc. (Cergy Pontoise, France). [
3
H]Myo-
inositol (23.4 Ci/mol) was purchased from PerkinElmer Life Sciences
(Paris, France). All other reagents used were of molecular or analytical
grade, where appropriate.
Phylogenetic analysis. Sequence alignment of the VFTMs of crystallized
amide-binding protein AmiC (pdb:1pea), leucine–isoleucine–valine-
binding protein (pdb: 2liv), natriuretic peptide receptors A and C (pdb:
1dp4 and 1jdn, respectively), and mGlu1 (pdb:1ewk) was deduced after
superimposition of their structures using SwissPdbViewer (version 3.7)
(Guex and Peitsch, 1997). Sequences of VFTMs of the rat NR2A subunit
of the NMDA receptor, GABA
B1
and GABA
B2
subunits (GB1 and GB2),
and tyrosine kinase receptor type 1 (RTK1) from Schistosoma mansoni
were aligned on the structural multiple alignment according to Paoletti et
al. (2000), Kniazeff et al. (2002), and Vicogne et al. (2003), respectively.
The phylogenetic tree was then constructed using the neighbor-joining
method (Saitou and Nei, 1987) with the command interface of the
Clustal W 1.60 program (Thompson et al., 1994) using the default pa-
rameters and not excluding gaps. Bootstrap values were calculated using
1000 trials with seeds of 111. The tree was drawn using TreeView (version
1.6.2) (Page, 1996).
Molecular modeling. Three-dimensional models of the open and closed
forms of GABA
B1
VFTM were built as described previously (Kniazeff et
al., 2002) using Modeler 6.0
␣
(Sali and Blundell, 1993). In silico mu-
tagenesis and disulphide bridges modeling were performed using the
SwissPdbViewer program (version 3.7) (Guex and Peitsch, 1997) and
software default parameters. The figures were prepared using
SwissPdbViewer.
Plasmids and site-directed mutagenesis. The plasmids encoding the
wild-type GABA
B1a
and GABA
B2
subunits epitope tagged at their
N-terminal ends (pRK-GABA
B1a
-HA and pRK-GABA
B2
-cMyc), under
the control of a cytomegalovirus promoter, were described previously
(Galvez et al., 2001; Pagano et al., 2001).
Mutant subunits, carrying single or multiple mutations, were obtained
using the Quick-Change strategy (Stratagene, La Jolla, CA). Briefly, for
each mutagenesis, two complementary 27-mers primers (Eurogentec,
Bruxelles, Belgium) were designed to contain the desired mutation. To
allow a rapid screening of mutated clones, primers carried an additional
silent mutation introducing a new restriction site. The presence of the
desired mutations and the absence of additional ones were confirmed by
DNA sequencing (Genaxis, Nıˆmes, France). For double mutants, two
Quick-Change reactions were performed successively.
Cell culture and expression in HEK 293. Human embryonic kidney
(HEK) 293 cells were cultured in DMEM supplemented with 10% FCS
and transfected by electroporation as described previously (Brabet et al.,
1998; Franek et al., 1999). Unless stated otherwise, 10 ⫻10
6
cells were
transfected with plasmid DNA containing hemagglutinin (HA)-tagged
GABA
B1
(2
␮
g) and cMyc-tagged GABA
B2
(2
␮
g) and were completed to
a total amount of 10
␮
g of plasmid DNA with pRK
6
. For determination of
inositol phosphate (IP) accumulation, the cells were also transfected with
the chimeric G
␣
qi9 G-protein, which allows the coupling of the recom-
binant heteromeric GABA
B
receptor to phospholipase C (PLC) (Franek
et al., 1999).
Determination of IP accumulation. Determination of IP accumulation
in transfected cells was performed in 96-well plates (0.2.10
6
cells/well)
after overnight labeling with
3
H-myo-inositols (0.5
␮
Ci/well), as de-
scribed previously (Chengalvala et al., 1999), with some modifications.
The stimulation was conducted for 30 min in a medium containing 10
mMLiCl and the indicated concentration of agonist or antagonist. The
reaction was stopped by replacing the medium with 0.1 Mformic acid.
Supernatants were recovered, and IP was purified by ion exchange chro-
matography using DOWEX AG1-X8 resin (Biorad, Marnes-la-Coquette,
France) in 96-well filter plates (ref:MAHVN4550; Millipore, Bedford,
MA). Total radioactivity remaining in the membrane fractions was
counted after treatment of cells with a solution containing 10% Triton
X-100 and 0.1N NaOH. Radioactivity was quantified using Wallac 1450
MicroBeta liquid scintillation counter. Data were expressed as the
amount of total IPs produced over the amount of radioactivity remaining
in the membranes plus the produced IP. Unless stated otherwise, all
data are means ⫾SEM of at least three independent experiments and
expressed in percentage of wild-type activity after a 30 min applica-
tion of 1 mMGABA. The dose–response curves were fitted using the
Figure1. Phylogenic analysis ofvarious VFTMs ( a) and comparison of the inactive and active
conformations of mGlu1 ( b) and NPRC ( c) VFTM dimers. a, The phylogenetic tree was con-
structed using the sequences of the VFTMs of the mGlu1 receptor, the amide-binding protein
(AmiC) from the amidase operon, the NR2A subunit of the rat NMDA receptor, leucine–
isoleucine–valine-bindingprotein(LIVBP),thenatriureticpeptidereceptortypesAandC(NPRA
and NPRC, respectively), RTK1 from Schistosoma mansoni, and the rat GABA
B1
and GABA
B2
subunits. Only branches with bootstrap values ⬎600 are shown. Note that the RTK1 VFTM is
closer to the GABA
B
VFTMs than to any other VFTMs. b, Ribbon view of the inactive (left; pdb:
1ewt) and active (right; pdb:1ewk) forms of the dimer of mGlu1 VFTMs. Note the change in the
relative orientation between the two VFTMs and the closure of one VFTM (the blue– green one
in front) that bring together lobe II. c, Ribbon view of the inactive (left; pdb:1jdn) and active
(right; pdb:1jdp) forms of the dimer of NPRC VFTMs. Note that ANP binding at the interface
between the two VFTMs brings together lobe II.
Kniazeff et al. •Locking the GABA
B
Receptor in Its Active State J. Neurosci., January 14, 2004 •24(2):370 –377 • 371

Graph Pad (San Diego, CA) Prism program and the following equation:
y⫽[(y
max
⫺y
min
)/(1 ⫹(x/EC
50
)
nH
)] ⫹y
min
, where the EC
50
is the
concentration of the compound necessary to obtain 50% of the maximal
effect, and nH is the Hill coefficient.
Ligand-binding assay. Ligand-binding assay on intact HEK 293 cells
was performed as described previously using 0.1 nM[
125
I]CGP64213
(Galvez et al., 2001). Displacement curves were performed with at least
seven different concentrations of the displacer, and the curves were fitted
according to the equation: y ⫽[(y
max
⫺y
min
)/(1 ⫹(x/IC
50
)
nH
)] ⫹y
min,
where the IC
50
is the concentration of the compound that inhibits 50% of
bound radioligand, and nH is the Hill coefficient. K
i
values were calcu-
lated according to the equation IC
50
⫽K
i
(1 ⫹[RL]/ K
d
), where [RL] and
K
d
are the concentration and dissociation constant of the radioligand,
respectively. K
d
was determined assuming K
i
⫽K
d
in the case of
CGP64213.
Western Blotting. Western blotting was performed as described previ-
ously (Kniazeff et al., 2002) using the rabbit polyclonal anti-HA antibody
(Zymed, San Francisco, CA) and the anti-rabbit HRP antibody (Amer-
sham, Saclay, France). The signal was revealed using an ECL assay.
Anti-HA ELISA assay for quantification of cell surface expression.
Twenty-four hours after transfection [10 ⫻10
6
cells, HA-tagged
GABA
B1
(2
␮
g), and cMyc-tagged GABA
B2
(2
␮
g) subunits], cells were
fixed with 4% paraformaldehyde and then blocked with PBS plus 5%
FBS. After a 30 min reaction with primary antibody (monoclonal
anti-HA clone 3F10; 0.5
␮
g/ml; Roche, Basel, Switzerland) in the same
buffer, the goat anti-rat antibody coupled with HRP (Jackson Immu-
noresearch, West Grove, PA) was applied for 30 min at 1
␮
g/ml. After
intense washes with PBS, secondary antibody was detected and quanti-
fied instantaneously by chemiluminescence using Supersignal ELISA
femto maximum sensitivity substrate (Pierce, Rockford, IL) and a Vic-
tor
2
luminescence counter (Wallac, Turku, Finland).
GTP-
␥
-
35
S binding measurements. Cells were transfected using Poly-
Fect transfection reagent (Qiagen, Hilden, Germany) under optimized
conditions. Complexes were formed using a total amount of 8
␮
gof
plasmid DNA with 60
␮
l of PolyFect in 300
␮
l of serum-free antibiotic-
free DMEM for 10 min and then added to cells at 40–60% confluency.
According to expression results, the amount of DNA is 2
␮
g of GABA
B1
,
1
␮
g of GABA
B2
,2
␮
gofG
␣
o1c, and 3
␮
gofpRK
6
for wild-type receptor
and 2
␮
g of GABA
B1
,2
␮
g of GABA
B2
,2
␮
gofG
␣
o1c, and 2
␮
gofpRK
6
for CC1 and CC2 mutants.
Forty-eight hours after transfection, cells were scraped in lysis buffer
(15 mMTris, 2 mMMgCl2, and 0.3 mMEDTA, pH 7.4) and centrifuged
twice. The pellet was resuspended in Tris (50 mM), MgCl2 (3 mM), EGTA
(0.2 mM), and NaCl (60 mM), pH 7.4, using a potter. GTP-
␥
-[
35
S] (1099
Ci/mmol; Amersham, Little Chalfont, UK) binding was performed in
96-well filtration plates (ref:MAFCN0B50; Millipore) equilibrated with
Tris (50 mM) and MgCl2 (5 mM), pH7.4. Membranes (5
␮
g/well) were
preincubated or not with GABA (15 min; 1 mM; final volume, 20
␮
l). The
plate was incubated for 1 hr at 30°C after the addition of 60
␮
l of incu-
bation buffer (50 mMTris, 1 mMEDTA, 10
␮
MGDP, 5 mMMgCl2, 0.01
mg/ml leupeptine, 100 mMNaCl, and 0.4 nMGTP-
␥
-[
35
S]) and 20
␮
lof
H
2
O. After vacuum filtration, plate-filter washing (three times with 250
␮
lof50mMTris), and drying, the radioactivity was measured using a
1450 MicroBeta liquid scintillation counter (Wallac).
Results
Double cysteine mutations expected to lock the GABA
B1
VFTM in a closed state generate constitutively
active receptors
Based on three-dimensional models of the open and closed forms
of the GABA
B1
VFTM (Galvez et al., 1999, 2000a; Kniazeff et al.,
2002), some positions were selected to introduce cysteines that
could form a disulphide bridge in the closed state only. As de-
picted in Figure 2, the side chains of Ser247 or Ser246 from lobe I
and those of Thr315 or Glu316 from lobe II are far apart in a
model of the open form (Fig. 2a) but are in close proximity in the
closed form (Fig. 2b,c). Indeed, in the double mutants S247C-
T315C (CC1) (Fig. 2d) and S246C-E316C (CC2) (data not
shown), the distance between the sulfur atoms in the closed form
models is compatible with the formation of a disulphide bond
(1.9 and 2.1 Åfor CC1 and CC2, respectively). Double cysteine
mutations were introduced in GABA
B1
, and the CC1 and CC2
mutants were analyzed for function after coexpression with wild-
type GABA
B2
. Expression of the CC mutants in HEK 293 cells led
to a high constitutive activity in the absence of agonist reaching
69.8 ⫾4.8% (CC1) and 48.8 ⫾4.3% (CC2) (n⬎3) of the max-
imal GABA-induced activity measured with the wild-type recep-
tor (Fig. 3a). In contrast, no significant increase in constitutive
activity can be detected with any of the single cysteine mutants
Figure 2. Three-dimensional models of the open ( a) and closed ( b) forms of the GABA
B1
VFTM and possible covalent linkage of both lobes in a closed form by a disulphide bridge. Lobe
I and lobe II are shown in red and blue ribbons, respectively. Residues subjected to mutagenesis
are represented. A closer view of these residues in the closed form model is shown in c.Ind, this
same region is shown for the CC1 mutant receptor, in which Ser247 and Thr315 were mutated
into cysteines.
Figure 3. The double cysteine mutants display a DTT-sensitive constitutive activity. Basal
andGABA (1 mM)-induced IP formation (black and white columns,respectively) weremeasured
in cells expressing wild-type (WT) or double cysteine GABA
B1
mutants, CC1 or CC2 ( a), or single
cysteine mutants S247C, T316C, and E316C ( b). c, Same as in a, but the GTP
␥
S binding was
measured in membranes prepared from cells expressing the indicated GABA
B1
subunit, the
wild-type GABA
B2
subunit, and the Go1
␣
subunit. d, Same as in a, but after a 30 min treatment
with 10 mMDTT, pH 8. Data are expressed as the percentage of the response obtained with the
wild-type receptor after 1 mMGABA stimulation and are means ⫾SEM of three independent
experiments (a,b,d) performed in triplicate. In c, data are means ⫾SEM of triplicate determi-
nations from one typical experiment.
372 •J. Neurosci., January 14, 2004 •24(2):370 –377 Kniazeff et al. •Locking the GABA
B
Receptor in Its Active State

(Fig. 3b). Such a property of the CC mutants can be detected
using either the artificial coupling of the GABA
B
receptor to PLC
with the chimeric Gqi9 protein (Fig. 3a), but also through the
direct activation of Go protein as shown by GTP
␥
[
35
S] binding
(Fig. 3c).
A disulphide bridge is involved in the constitutive activity of
CC mutants
In agreement with the involvement of a disulphide bond in the
high constitutive activity of CC mutants, this activity can no
longer be detected after reduction with DTT (Fig. 3d). This later
effect does not result from a destabilization of any of the subunits
attributable to reduction of native disulphide bonds for two rea-
sons. First, DTT treatment of cells expressing the wild-type re-
ceptor had no significant effect on the basal activity, and only a
small decrease in GABA-mediated response was observed (Fig.
3d). Second, after reduction, CC mutants can still be activated by
agonists (Fig. 3d) with an affinity similar to that measured on the
wild-type receptor (Table 1).
The constitutive activity of CC mutants is not inhibited by
competitive antagonists
Competitive antagonists of mGluRs have been shown to bind in
the open form of the VFTM, as illustrated with the mGlu1
(Tsuchiya et al., 2002) and mGlu8 (Bessis et al., 2002) receptors.
Accordingly, a receptor locked in a closed state would not be
expected to bind competitive antagonists. As shown in Figure 4a,
the antagonist CGP64213 did not inhibit the basal activity of the
CC mutants, whereas it was able to (1) inhibit the basal activity of
the wild-type receptor and (2) antagonize GABA-mediated re-
sponses of single cysteine mutants (Table 1). Moreover, after
DTT treatment, this antagonist become effective in fully inhibit-
ing GABA activation of the CC mutants (Fig. 4b) demonstrating
that a disulphide bridge, but not the presence of the two cysteines,
is responsible for the absence of antagonist action. These data
show that the constitutively active receptor is locked in its active
state and cannot return to the inactive antagonist-stabilized state.
Not all surface-expressed CC1 mutants are locked in an
active state
Although high constitutive activity can be observed in cells ex-
pressing the CC1 mutant, GABA [as well as its chlorophenyl de-
rivative baclofen (data not shown)] could still further increase the
response with a potency identical to that measured on the wild-
type receptor (Fig. 5a). This could result from the disulphide
bridge-stabilized receptors being in a partially active conforma-
tion. However, the agonist-induced response can be fully antag-
onized by CGP64213 with the same potency as that measured on
the wild-type receptor (Fig. 5b), indicating that the antagonist has
access to agonist-activated receptors. These data suggest that
there are two populations of receptors, some constitutively active
and insensitive to antagonists, and some that can be activated by
agonists and inhibited by CGP64213. In agreement with this pro-
posal, a small amount of specific [
125
I]CGP64213 binding can be
detected in cells expressing the CC1 mutant, and this binding was
largely increased after DTT treatment (approximately fivefold) in
contrast to the wild-type receptor (Fig. 6a). This effect was not
attributable to a change in the CGP64213 affinity because the
same K
i
values for this antagonist could be determined by dis-
placement studies on CC1-expressing cells before and after DTT
treatment (Table 1). This shows that reduction reveals new bind-
Table 1. Potencies of the agonist GABA and the antagonist CGP64213 on wild-type (WT), CC1, CC2, and the single cysteine mutants measured either with a functional assay
(IP production) or by displacement of [
125
I]CGP64213 binding under control condition or after DTT treatment
IP production [
125
I]CGP64213 binding
Control DTT Control DTT
GABA
EC
50
(
␮
M)
CGP64213
IC
50
(nM)
GABA
EC
50
(
␮
M)
CGP64213
IC
50
(nM)
GABA
K
i
(
␮
M)
CGP64213
K
i
(nM)
GABA
K
i
(
␮
M)
CGP64213
K
i
(nM)
WT 0.23 ⫾0.02 10.5 ⫾1.3 0.34 ⫾0.06 12.6 ⫾0.8 4.74 ⫾0.61 1.28 ⫾0.13 2.75 ⫾0.97 1.53 ⫾0.21
CC1 0.43 ⫾0.07 13.1 ⫾2.7 0.26 ⫾0.08 10.3 ⫾0.3 n.t. 3.61 ⫾0.81 1.59 ⫾0.34 3.43 ⫾1.00
CC2 0.33 ⫾0.07 12.8 ⫾0.7 0.29 ⫾0.05 12.0 ⫾0.7 n.t. 2.80 ⫾0.96 n.t. n.t.
S247C 0.36 ⫾0.06 n.t. n.t. n.t. 8.41 ⫾0.80 1.67 ⫾0.73 n.t. n.t.
T315C 0.28 ⫾0.06 n.t. n.t. n.t. 9.50 ⫾0.46 1.88 ⫾0.98 n.t. n.t.
E316C 0.85 ⫾0.017 n.t. n.t. n.t. 5.92 ⫾1.5 2.13 ⫾0.73 n.t. n.t.
Values are means ⫾SEM of at least three independent experiments. n.t., Not tested.
Figure 4. Constitutive activity of CC mutants is not inhibited by the GABA
B
competitive
antagonist CGP64213. a, The high basal IP production measured with the CC mutants (black
columns) is not inhibited by the GABA
B
antagonist CGP64213 (300 nM; gray columns), in con-
trast to the basal activity of the wild-type (WT) receptor. b, CGP64213 antagonizes with the
same potency GABA (10
␮
M)-stimulated IP formation in cells expressing the wild-type GABA
B
receptor under control condition (black circles) or after DTT treatment (gray circles), or in cells
expressing the CC1 mutant after DTT treatment (gray squares). Data are means ⫾SEM of
triplicates from one representative of three independent experiments.
Figure 5. Cells expressing CC1 remain sensitive to both GABA and CGP64213 with the same
potency as cells expressing wild-type (WT) receptor. Concentration-dependent effect of GABA
(a) or CGP64213 (in the presence of 10
␮
MGABA) ( b) on IP production in cells expressing
wild-type (circles, dashed line) or CC1 (squares, solid line) GABA
B
receptor. Data are means ⫾
SEM of triplicates from one representative of three independent experiments.
Kniazeff et al. •Locking the GABA
B
Receptor in Its Active State J. Neurosci., January 14, 2004 •24(2):370 –377 • 373

ing sites in cells expressing the CC1 mutant. Finally, the specific
binding of [
125
I]CGP64213 after DTT treatment represented half
of that determined on cells expressing the wild-type receptor, in
agreement with a twofold lower expression level of this receptor
(Fig. 6b,c). Therefore, most CGP64213-binding sites have been
unmasked after DTT reduction. Taken together, these data show
that 75.5 ⫾6.9% of the binding sites in cells expressing the CC1
mutant are inaccessible to CGP64213 because of a disulphide
bridge. Thus, the effect of GABA on these cells likely results from
the 25% remaining receptors because this effect is fully inhibited
by this antagonist. Another conclusion from these data are that
GABA is unlikely able to activate the constitutively active,
CGP64213-insensitive form of the receptor.
We then examined whether the proportion of disulphide-
locked receptors could be modulated by oxidizing agents 5,5⬘-
dithio-bis(2-nitrobenzoic acid) or Cu-Phenantroline by agonists
that are expected to stabilize the closed state or by antagonists
expected to prevent the formation of the closed state. None of
these treatments alone or in combination lead to a modification
of constitutive activity (Fig. 7). Moreover, after DTT treatment,
constitutive activity of the CC1 mutant could not be restored
after 30 min of exposure to the oxidizing agents (data not shown).
This suggests that all disulphide-locked receptors are formed in-
side the cells rather than after plasma membrane insertion.
Constitutive activity of the disulphide-locked CC1 mutant is
close to the agonist-induced activity of the wild-type receptor
We then examined whether the constitutive activity of the CC1
mutant corresponds to a fully active form of the receptor. Surface
expression was determined with an ELISA assay, and second mes-
senger formation was measured in cells expressing various
amount of the GABA
B
heterodimer. A saturating curve was ob-
served, and the slope at the origin of the curves is indicative of the
specific activity of the receptor (amount of second messengers
produced per receptor) (Fig. 8). In the absence of agonist, the
specific activity of CC1 is about half the specific activity of the
wild-type receptor activated by GABA (0.7 ⫻10
⫺3
and 1.4 ⫻
10
⫺3
arbitrary units, respectively), but, after DTT treatment, this
activity is decreased to the level of the basal activity of the wild-
type receptor. Because only 75% of the CC1 receptors are ex-
pected to be stabilized in an active form by a disulphide bridge
(see above), this indicates that the specific activity of the consti-
tutively active form of CC1 receptors is close to the specific activ-
ity of the agonist-activated wild-type receptors (1.0 ⫻10
⫺3
and
1.4 ⫻10
⫺3
arbitrary units, respectively).
The curves obtained clearly saturate, indicating a maximal IP
formation could be measured with a nonsaturating amount of
receptors expressed at the cell surface. This is in contrast to what
can be observed under similar conditions with the Gq-coupled
mGlu5 receptor (Goudet et al., 2004). This may, therefore, result
from the use of the chimeric G-protein
␣
subunit Gqi9 cotrans-
fected with the receptor to make it able to activate PLC. It is
possible that either the amount of Gqi9 or endogenous
␤␥
sub-
units is limiting when the receptor is overexpressed.
Double cysteine mutations in the GABA
B2
VFTM does not
influence receptor function
Can the closure of the GABA
B2
VFTM also lead to receptor acti-
vation? To test this possibility, cysteines were introduced in the
GABA
B2
VFTM at the same positions as in GABA
B1
, but neither
constitutive activity nor changes in agonist efficacy and potency
were detected (data not shown). To examine whether such cys-
teines could form a disulphide bond in the GABA
B2
VFTM, and
as such to lock this domain in a closed state, Western blot exper-
iments were conducted under reducing and nonreducing (100
mMDTT, pH 8) conditions. Such an experiment performed with
the full-length receptor did not lead to a clear conclusion, likely
because of the large size of these receptor subunits. As such, con-
structs were created (⌬GB1 and ⌬GB2), in which a stop codon
Figure 6. Cells expressing CC1 GABA
B
receptors possess CGP64213-binding sites at their
surface, and new sites are unmasked after DTT treatment. a,[
125
I]CGP64213 binding under
control condition (white column) or after DTT treatment (black column) on intact cells express-
ing GABA
B2
and wild-type (WT) or CC1 GABA
B1
subunits. Data are means of triplicates from one
representative of three independent experiments. b, Western blot on total membranes of cells
mock-transfected or transfected with plasmid expressing HA-tagged wild-type or CC1 mutant.
c, Quantification of cell surface expression of wild-type (WT) and CC1 mutant GABA
B1
subunit
using an ELISA assay in HEK 293-transfected cells.
Figure 7. Neither oxidizing treatment nor overnight incubation with agonist or antagonist
increasetheconstitutive activity. Basal and GABA (1 mM)-induced IP formation (black and white
columns, respectively) were measured under control (CTR) or after various treatment: 0.5 mM
5,5⬘-dithio-bis(2-nitrobenzoic acid (DTNB) for 30 min, 1 mMCuSO
4
plus 4 mMphenanthroline
(CuP) for 30 min, CuP in the presence of 10
␮
MGABA for 30 min, and overnight incubation with
CGP64213 (100 nM) or GABA (1 mM) applied immediately after transfection.
374 •J. Neurosci., January 14, 2004 •24(2):370 –377 Kniazeff et al. •Locking the GABA
B
Receptor in Its Active State

was introduced after TM1, leading to the expression of the VFTM
attached to the membrane by the first transmembrane domain.
As shown in Figure 9, these constructs migrated faster under
nonreducing conditions, in agreement with the proposed exis-
tence of native disulphide bridges in the VFTM of both GABA
B1
and GABA
B2
(Galvez et al., 2000a; Kniazeff et al., 2002). Of inter-
est, both ⌬GB1-CC and ⌬GB2-CC migrated slightly faster than
the wild types under the nonreducing condition, consistent with
the existence of an additional disulphide bridge in these
constructs.
Discussion
The present study indicates that the introduction of two cysteines
expected to lock the GABA
B1
VFTM in a closed form generates a
constitutively active heterodimeric GABA
B
receptor.
A disulphide bond locks the GABA
B
receptor in an active state
GPCRs are assumed to oscillate between various inactive (R) and
active (R*) conformations (Lefkowitz, 1993; Samama et al., 1993;
Leff, 1995; Farrens et al., 1996; Christopoulos and Kenakin,
2002), the latter being stabilized by agonists, whereas the former
is stabilized by inverse agonists. Mutations have been identified
that lead to constitutive activity (Lefkowitz, 1993; Samama et al.,
1993; Leff, 1995; Christopoulos and Kenakin, 2002; Parnot et al.,
2002), but these have been shown to destabilize the inactive con-
formations rather than to stabilize an active one (Parnot et al.,
2002). As such, these mutations are expected to displace the
R7R* equilibrium toward R*, as demonstrated by the inhibition
of constitutive activity by inverse agonists, but did not bring
much information on the active conformation of the receptor.
The GABA
B1
CC mutants appear to be locked in an active state
through the formation of an additional disulphide bond. Indeed,
the R7R* equilibrium seems to no longer exist because the re-
ceptor cannot return to the inactive state even in the presence of
a high concentration of the inverse agonist CGP64213. Only DTT
treatment allowed the receptor to return to an inactive state. This
represents the first example of a constitutively active form of a
GPCR resulting from the locking of an active state.
A disulphide bond locks the GABA
B1
VFTM in a closed state
Several data are consistent with the two cysteines introduced in
the CC1 and CC2 mutants involved in a disulphide bridge that
locks the GABA
B1
VFTM in a closed state. First, the VFTM of the
CC1 mutant migrates faster than the wild type in acrylamide gels
under nonreducing conditions. Second, the distance between the
two sulfydryl groups introduced is consistent with the formation
of a disulphide bond in a closed form model of the GABA
B1
VFTM only. Third, the GABA
B
antagonist CGP64213 is unable to
(1) inhibit the constitutive activity of the CC mutants and (2)
bind on the disulphide-locked receptor, although this antagonist
can bind and exert its normal antagonist action after DTT treat-
ment. Indeed, as observed for the mGlu1 (Tsuchiya et al., 2002)
and mGlu8 (Bessis et al., 2002) receptors, many GABA
B
compet-
itive antagonists are larger than agonists and cannot fit into a
closed form model of the binding site. Finally, modeling and
mutagenesis studies suggested that the active form of the GABA
B1
VFTM corresponds to a closed form because residues from both
lobes are involved in agonist binding (Galvez et al., 1999, 2000a;
Bernard et al., 2001; Costantino et al., 2001; Kniazeff et al., 2002).
However, the absence of modulation by oxidants or ligands
expected to help or prevent the formation of the disulphide
bridge was surprising. Accordingly, the disulphide-locked recep-
tors are likely formed during the synthesis of the receptor in the
endoplasmic reticulum. When the receptor is correctly folded
and at the cell surface, in the absence of the preformed disulphide
bond or after its reduction with DTT, the sulfydryl groups may
not be in a correct orientation to spontaneously form a disul-
phide bond. Indeed, in our three-dimensional model, the side
chains of mutated residues had to be reoriented manually for the
disulphide bridge to form. Moreover, this area in the GABA
B1
VFTM has been proposed to constitute a Ca
2⫹
-binding site
(Galvez et al., 2000b; Costantino et al., 2001), thus stabilizing a
precise position of the side chains of the surrounding residues
and possibly preventing the formation of a disulphide bond.
The disulphide-locked active state is close to the agonist-
stabilized active state
Although cells expressing the CC1 or CC2 mutants display a high
constitutive PLC activity, IP formation could still be increased by
Figure 8. Constitutive activity of CC1 mutant receptors is close to GABA-mediated activity of
the wild-type receptor. Basal (diamonds) and GABA-mediated (squares) IP formation were
measured in cells transfected with HA-tagged wild-type (open symbols, thin lines) or CC1 mu-
tant (filled symbols, bold lines) GABA
B1
subunit (2
␮
g) and various amounts of GABA
B2
-
expressing plasmids (0.05–2
␮
g). The amount of GABA
B
receptors at the cell surface was
measuredusingELISAandaHAantibody.BasalIPformationincellsexpressing various amounts
of CC1 mutant containing GABA
B
receptor was also measured after DTT treatment (closed tri-
angles). Data are means ⫾SEM of triplicate determinations from a single experiment. Similar
data were obtained in three independent experiments. Data points were fitted according to the
saturation equation: IP ⫽IPmax ⫻Exp/(Cte ⫹Exp), where IP is the amount of IPs produced
under the indicated condition, IPmax is the maximal IP response obtained with a high expres-
sion level of the receptor, Exp is the signal obtained with the ELISA, and Cte is a constant.
Figure 9. Migration of the VFTMs of GABA
B1
and GABA
B2
is affected by the presence of the
two additional cysteines. Total proteins from cells expressing the wild-type (WT) or the CC
version of ⌬GB1 or ⌬GB2 (GABA
B1
and GABA
B2
, in which a stop codon has been introduced
after TM1) were separated on an acrylamide gel under nonreducing conditions or after treat-
ment with 100 mMDTT, pH 8. After transfer, the proteins were detected using an anti-HA
antibody directed against the HA epitope inserted after the signal peptide. Data shown are
representative of three independent experiments.
Kniazeff et al. •Locking the GABA
B
Receptor in Its Active State J. Neurosci., January 14, 2004 •24(2):370 –377 • 375

GABA
B
agonists. This may seem surprising in the case of the CC2
mutant because Ser246 (mutated into Cys in this mutant) has
been proposed previously to form an H-bond with the carboxylic
function of GABA
B
ligands (Galvez et al., 2000a; Bernard et al.,
2001; Costantino et al., 2001; Kniazeff et al., 2002). However,
because an SH group can also form H-bonds, it is likely that the
replacement of the OH group of Ser246 by an SH group has
minor effect on ligand-binding affinities.
In the case of the ionotropic glutamate receptor subtypes,
partial agonists stabilized a partially closed form of the
glutamate-binding domain (Armstrong and Gouaux, 2000; Arm-
strong et al., 2003). Therefore, one possibility to explain the
agonist-induced response in CC mutant-expressing cells was that
the disulphide bond locked the receptor in a partially closed form
that could be closed further in the presence of agonist. However,
our data indicate that this is unlikely the case but rather that the
agonist-mediated response is attributable to a certain fraction of
the receptors at the cell surface that are not locked in their active
state by a disulphide bridge. Indeed, a fraction of the CC mutants
can bind with a normal affinity to the antagonist CGP64213, and
the agonist-induced activity can be inhibited by the antagonist
with a wild-type K
i
. Taking this into consideration, together with
the receptor density at the cell surface, we estimated that the
specific activity of the constitutively active disulphide-locked
CC1 mutant is close to that of the agonist-stabilized form of the
wild-type receptor. This suggests that the conformation of the
disulphide-locked CC1 VFTM is close to that of the agonist-
bound form of the wild-type receptor.
Comparison with mGlu1 and ANP receptors
As shown in Figure 1a, the GABA
B
VFTMs are as distant from the
mGlu1 VFTM than from any other VFTMs. Moreover, the large
insertions found in mGlu VFTMs and conserved in the related
Ca
2⫹
-sensing, pheromone, and taste receptors are not found in
the GABA
B
VFTMs. Finally, whereas the mGlu-like receptors
possess a cysteine-rich domain that connects their VFTM to the
heptahelical domain, the GABA
B
subunits do not. In contrast,
the GABA
B
VFTMs share a significant higher similarity with the
VFTM of the monotopic receptor RTK1 (Vicogne et al., 2003),
leaving open the possibility that the dimer of VFTMs of the
dimeric GABA
B
receptor functions like those of dimeric mono-
topic receptors. However, in the case of the monotopic receptor
for the natriuretic peptide, both VFTMs are in a closed confor-
mation even in the inactive state (He et al., 2001; van den Akker,
2001) (Fig. 1c), whereas agonists seem to stabilize a closed form of
the VFTM in the case of the mGluRs (Kunishima et al., 2000;
Bessis et al., 2002; Tsuchiya et al., 2002) (Fig. 1b). Accordingly,
our data suggest the dimer of GABA
B
VFTMs functions like that
of mGluRs despite some structural dissimilarities between these
two types of receptors.
Role of GABA
B2
VFTM in GABA
B
receptor activation
GABA
B2
possesses a VFTM similar to that of GABA
B1
. Although
required for GABA
B
receptor activation (Galvez et al., 2001;
Margeta-Mitrovic et al., 2001a), the GABA
B2
VFTM does not
seem to bind any ligand (Kniazeff et al., 2002). Our data show
that introduction of two cysteines at the same position as in CC1
and CC2 mutants in the GABA
B2
VFTM does not change the
properties of the heteromeric GABA
B
receptor. However, as ob-
served with the GABA
B1
CC mutant, the GABA
B2
CC migrates
faster than the wild type under nonreducing conditions, consis-
tent with the existence of an additional disulphide bridge in this
mutant. This suggests that the closure of GABA
B2
is not sufficient
to activate the receptor. Indeed, either GABA
B2
VFTM is always
closed or the closure of GABA
B2
VFTM has no detectable effect
on the function of the receptor. This is consistent with the closure
of GABA
B1
VFTM being sufficient for GABA
B
receptor activa-
tion, in agreement with our proposal that GABA
B
agonists bind
in the GABA
B1
VFTM only (Kniazeff et al., 2002). Moreover, our
data also suggest that even an artificial ligand interacting in the
cleft of GABA
B2
is not expected to have an important effect on the
functioning of the heterodimer.
Allostery between the four main domains of the
heterodimeric GABA
B
receptor
Although GABA binds to the GABA
B1
VFTM only, the GABA
B2
7TM is critical for G-protein coupling (Galvez et al., 2001;
Margeta-Mitrovic et al., 2001b; Robbins et al., 2001; Duthey et al.,
2002). How can the closure of the GABA
B1
VFTM lead to the
change in conformation of the GABA
B2
7TM domain necessary
for G-protein activation? In the case of mGluRs, it has been pro-
posed that the change in the relative orientation of the two
VFTMs, observed on glutamate binding, may be required for
activation (Kunishima et al., 2000; Jensen et al., 2001, 2002).
Based on this hypothesis, the closure of the GABA
B1
VFTM pos-
sibly leads to a change in the relative orientation of the two
VFTMs in the heterodimer as proposed for the mGlu1 receptor,
no matter whether the GABA
B2
VFTM is in a closed or open
conformation.
Conclusion
Our present study brings much information on how GABA acti-
vates the GABA
B
receptor and on the specific roles played by each
subunit in this heteromeric receptor. Moreover, such informa-
tion may be of interest for the understanding of the activation
process of many other GPCRs, Indeed, the rhodopsin-like class I
GPCRs also form dimers (Bouvier, 2001; Fotiadis et al., 2003). It
is still unknown whether such dimers are required for activation
and whether, in such a case, agonist occupation of a single pro-
tomer is sufficient for full activation of the receptor. Taking into
account our data and the fact that retina can detect a single pho-
ton although rhodopsin forms oligomers of dimers (Fotiadis et
al., 2003; Liang et al., 2003), one may propose that an initial
change in conformation in a single subunit may be sufficient to
trigger the activation of some dimeric GPCRs.
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