mapping the agonist binding site of gabab type 1 subunit sheds

Mapping the Agonist Binding Site of GABAB Type 1 subunit sheds light on the activation process of GABAB receptors*

Thierry Galvez ‡†, Laurent Prézeau ‡, Gérald Milioti ‡, Miloslav Franek ‡#, Cécile Joly ‡,

Wolfgang Froestl ¶, Bernhard Bettler ¶, Hugues-Olivier Bertrand §, Jaroslav Blahos ‡#, and

Jean-Philippe Pin ‡

‡ Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141

rue de la Cardonille, F-34094 Montpellier, France

# Laboratory of Molecular Physiology, Charles University 3rd Medical School and Czech

Academy of Sciences, Ke Karlovu 4, Prague 2, Czech Republic

¶ TA Nervous system, Novartis Pharma AG, CH-4002 Basel, Switzerland

§ Molecular Simulations Inc., Parc Club Orsay Université, 20 rue Jean Rostand, 91893 Orsay

Cedex, France

Running title: The agonist binding pocket of the GABAB1 subunit

† To whom correspondence should be addressed:

Thierry GALVEZ

CNRS-UPR9023

CCIPE, 141 rue de la Cardonille

F-34094 Montpellier Cedex 5 - France

Tel: +33 467 14 2933

Fax: +33 467 54 2432

e.mail: [email protected]

* This work was supported by grants from the CNRS, the French Ministry of Education and

Research (Programme Physique et Chimie du Vivant), the European Community (Biotech2

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 13, 2000 as Manuscript M007848200 by guest on January 31, 2018

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SUMMARY

The γ-amino-n-butyric acid type B (GABAB) receptor is constituted of two subunits,

GABAB1 and GABAB2, belonging to the family-3 heptahelix receptors. These proteins

possess two domains : a seven transmembrane core and an extracellular domain containing the

agonist binding site. This binding domain is likely to fold like bacterial periplasmic binding

proteins which are constituted of two lobes that close upon ligand binding. Here, using molecular

modeling and site directed mutagenesis, we have identified residues in the GABAB1 subunit

which are critical for agonist binding and activation of the heteromeric receptor. Our data

suggest that two residues (Ser246, Asp471) located within lobe-I form H-bonds and a salt

bridge with carboxylic and amino groups of GABA respectively, demonstrating the pivotal role

of lobe-I in agonist binding. Interestingly, our data also suggest that a residue within lobe-II

(Tyr366) interacts with the agonists in a closed form model of the binding domain, and its

mutation into Ala converts the agonist baclofen into an antagonist. These data demonstrate the

pivotal role played by the GABAB1 subunit in the activation of the heteromeric GABAB

receptor and are consistent with the idea that a closed state of the binding domain of family 3

receptors is required for their activation.

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INTRODUCTION

The γ-amino-n-butyric acid type B (GABAB1) receptor is a member of the family 3

heptahelix receptors (1). This protein family also includes the metabotropic glutamate (mGlu)

(2), the Ca2+-sensing (CaS) (3), fish olfactory (4) and some mammalian putative taste (5) and

pheromone (6, 7) receptors. An original feature of these receptors is that their ligand binding site

is located within their large extracellular amino-terminal domain structurally independent from

the transmembrane heptahelix core region (8-10). It is currently not known how the agonist

binding within this extracellular domain leads to the conformational changes of the heptahelical

core region required for G-protein activation (11). Answering this question will give insights

into the specific functional properties of these family 3 receptors. As a first step, it is important

to get information on the structural features of their ligand binding domain.

It has previously been proposed that the ligand binding domain of family 3 heptahelix

receptors displays a 3D structure similar to that of bacterial periplasmic binding proteins (PBP)

such as the Leucine/Isoleucine/Valine-binding protein (LIVBP) (12-15). These PBPs are part of

a family of structurally related proteins which also includes AmiC, a cytoplasmic amide binding

protein involved in the control of the amidase operon in P. aeruginosa (16, 17). These proteins

consist of two distinct globular lobes interconnected by three linkers (16, 18, 19). Numerous

structural studies have revealed that these PBPs adopt at least two stabilized conformations: an

“open”, usually unbound form and a “closed” form stabilized by the bound ligand (19). In the

closed conformation, the ligand is trapped between the two lobes. Accordingly it has been

proposed that agonist binding at family 3 receptors stabilizes an active (closed) conformation of

their binding domain which in turn activates the heptahelix core region (11).

In agreement with the above described hypothesis, the ligand binding domains of mGlu1 (8),

mGlu4 (9) and GABAB1 (10) receptors have been shown to retain their ligand binding

properties in the absence of the heptahelix domain. Moreover, molecular modeling and

mutagenesis studies have further confirmed the similarity between the ligand binding sites of

PBPs such as LIVBP and that of family 3 receptors (13, 14). Up to now, only residues within one

lobe (lobe-I) have been identified as being important for agonist binding on family 3 receptors

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(mGlu1, mGlu4 and CaS receptors) (13, 14, 20). Therefore the importance of the second lobe

(lobe-II) for ligand binding and receptor activation remains to be elucidated.

Another feature of family 3 heptahelix receptors is that they are found mostly as dimers (21,

22). The role of this dimerization process in the activation of the receptor is still unclear. Within

this receptor family, the GABAB receptor constitutes an excellent model for the study of the

activation mechanism of these receptors. Indeed, this receptor is an heterodimer constituted of

two subunits, GABAB1 and GABAB2, both necessary for a fully functional receptor (23-28).

Accordingly, the role of each subunit in the activation process can be analyzed.

In a previous study, we provided evidence supporting a similar 3D structure for LIVBP and

the binding domain of GABAB1 subunit, the only subunit able to bind the high affinity GABAB

receptor radioligands (24), and identified Ser246 as being critical for binding of the antagonist

[125I]-CGP64213 (15). In the present study, we have identified residues in the lobe-I of the

GABAB1 subunit critical for agonist binding and activation of the heteromeric receptor.

Moreover we show that the mutation of a residue in lobe-II of the GABAB1 subunit converts the

specific heteromeric GABAB receptor agonist baclofen, into an antagonist. In addition to

demonstrate the requirement of GABA binding on GABAB1 subunit for the activation of the

GABAB heteromeric receptor, our data are consistent with the idea that a closed form of the

binding domain of family 3 receptors is required for their activation.

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EXPERIMENTAL PROCEDURES

Materials - GABA was obtained from Sigma (L’Isle d’Abeau, France). [125I]-CGP64213

was synthesized as described elsewhere (29) and labeled to a specific radioactivity of >2,000 Ci

mmol-1 (ANAWA AG, Wangen, Switzerland). L-Baclofen was synthesized in the research

laboratories of Novartis Pharma in Basel (30). Serum, culture media, and other solutions used for

cell culture were from Life Technologies, Inc. (Cergy Pontoise, France).

Site directed mutagenesis - Single amino-acid substitution was carried out by the Quick

Change strategy (Stratagene, La Jolla, CA) according to the manufacturer’s instructions using

pBSB5 as a template (15). For each mutagenesis, two complementary 30-mers oligonucleotides

(sense and anti-sense; Genaxis Biotechnologie, Nimes, France) were designed to contain the

desired mutation in their center. 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 was subcloned in place of

the corresponding wild-type fragment of pRKBR1a (15).

Cell culture and expression in HEK293 cells - Human embryonic kidney (HEK) 293 cells

were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies SARL, Cergy-

Pontoise, France) supplemented with 10% fetal calf serum, penicillin and streptomycin. Wild

type and mutated expression constructs were transfected in HEK 293 cells by electroporation as

described previously (31). Electroporation was carried out in a total volume of 300 µl with

10x106 cells. For membrane preparation, 2 µg plasmid DNA containing either wild type or the mutated

GABAB1a receptor coding sequences and 8 µg of carrier DNA were used. After electroporation,

the cells were plated on polyornithine-coated dishes.

Ligand binding assay - Ligand competition experiments were performed on membranes of

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HEK-293 cells prepared as followed : 24h 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, 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 unlabelled ligands at the indicated concentration. The incubation

was terminated by filtration through GF/C Whatman glass fibre filters (Whatman International

Ltd., Maidstone, England). The concentration of [125I]-CGP64213 used in displacement

experiments (0.1 nM ) is approximately 10 times lower than the affinity of this radioligand on

the native or GABABR1 receptor, such that the IC50 values measured for all unlabeled

compounds are not significantly different from their affinity. Non-specific binding was

determined with 10 mM GABA. Displacement curves were fitted with the Kaleidagraph

software (Abelbeck software, USA) using the equation y=[(ymax-ymin)/(1+(x/IC50)nH))+ymin

where IC50 is the concentration of cold drug necessary to displace half of the specifically bound

[125I]-CGP64213 and nH is the Hill number.

Determination of inositol phosphate (IP) accumulation - HEK 293 cells were

transfected as described above with either wild-type or mutated GABAB1a receptor expressing

plasmids (pRKBR1a, 2 µg), GABAB2 receptor (pCI-Neo-BR2, 2 µg) (25), Gqi9 (2 µg) (32)

and carrier DNA (4µg). Determination of IP accumulation in transfected cells was performed 15h

after transfection and after overnight metabolic labeling of transfected cells with [3H]-myo-

inositol (23.4 Ci/mol, NEN, France). After three washes with Krebs buffer (NaCl 146 mM, KCl

4.2 mM, MgCl2 0.5 mM, Glucose 0.1 %, Hepes 20 mM/pH 7.4), the stimulation was conducted

for 30 min in Krebs buffer containing 10 mM LiCl and the indicated concentration of agonist.

The stimulation was stopped by replacement of the incubation medium with perchloric acid (5%)

on ice and the IPs were purified on Dowex AG 1-X8 (Biorad, Ivry sur Seine, France) columns.

Results are expressed as the amount of IP produced over the radioactivity present in the

membranes. Concentrations-response curves were fitted as described for the binding assay using

the equation y=[(ymax-ymin)/(1+(x/EC50)nH))+ymin where EC50 is the agonist concentration

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necessary to produce half of the maximal response.

Biotinylation of membrane proteins and western blotting - Cell surface proteins of

transfected HEK 293 cells were labeled with the membrane impermeant reagent Sulfo-NHS-

Biotin (Pierce, Bezons, France). Briefly, adherent cells were washed 3 times on ice with borate

buffer (10 mM H3BO3 pH 8.8, 140 mM NaCl) and incubated with 0.2 mg/ml of Sulfo-NHS-

Biotin in borate buffer for 30 min. The reaction was stopped by washing followed by incubation

in borate buffer with 100 mM NH4Cl for 10 min on ice. The cells were scraped in lysis buffer

(HEPES 20 mM pH 7.4, NaCl 100 mM, EDTA 5 mM) and the membranes pelleted and

solubilized in lysis buffer containing 1% Nonidet P40, 0.5% Na Deoxycholate and 0.1% SDS at

a final concentration of 5 mg/ml. The lysate was centrifuged for 1 h at 100,000 g. The soluble

fraction was incubated with streptavidin-coated agarose beads, overnight at 4°C. Bound proteins

were analyzed using Tricine-SDS-PAGE and immunoblotting using the polyclonal anti-

GABAB1 receptor antibody (33) and an ECL chemiluminescence system (Amersham Pharmacia

Biotech, Orsay, France). Analysis of wild-type or mutant receptor expression was conducted

according to the same protocol.

Homology modeling of open and closed conformations and docking of baclofen - To

generate the open form model of the GABAB1 receptor extracellular domain, we modified the

multiple alignment proposed in a previous work (15) in order to take into account the secondary

structure prediction of the GABAB1 receptor extracellular domain, as determined using the PHD

program (http://maple.bioc.columbia.edu/predictprotein/), and the sequence of the recently

cloned GABAB2 receptor. Compared to our previous alignment, this resulted in a change from

residue 467 to 486 containing helix αIX of LBP/LIVBP (18). The alignment of AmiC with

LIVBP and LBP was deduced from the structural superposition of each lobe of the closed form

of AmiC on the corresponding lobe of the open form of LIVBP (16).

The open form model of the GABAB1 receptor extracellular domain were generated by the

automated homology modeling tool MODELER 5.00 (InsightII version 980, MSI) (34) as

previously described (15). The closed form model was constructed using the coordinates of the

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two lobes of LBP (PDB code: 2lbp ; lobe-I: Glu 1-Arg 116, Pro 256-Asp 330; lobe-II: Ser

122-Thr 247) and LIVBP (PDB code: 2liv ; lobe-I: Glu 1- Arg 116, Pro 256-Asp 323; lobe-II:

Ser 122- Thr 247) crystalline open forms, the coordinates of the two lobes of the open form

model of GABAB1 receptor extracellular domain (lobe-I: Ser164-Arg284, Met277-Ser368

;lobe-II: Thr 127-Thr 261), and the coordinates LIVBP closed form model (generous gift from

Drs. F. Quiocho and P. O’Hara) as a template for the linkers of the hinge region. Similarly, AmiC

crystalline closed form (PDB code: 1pea) was used as a template. The resulting closed form

model of GABAB1 receptor was comparable to that obtained with the closed form model of

LIVBP. Only the latter has been used for the modeling experiments shown in this paper. The

3D/1D compatibility scores for these models (35), as determined using the Profiles_3D

algorithm using a sequence window of 21 amino acids (InsightII version 980, MSI), is always

positive and similar to that previously published (15). The global score is in the range of those

determined for the refined 3D structures of LIVBP, LBP or AmiC determined from X ray (data

not shown). Most residues of our models were in allowed regions of the Ramachandran’s map.

For “docking” experiments, baclofen was designed with a deprotonated carboxylic group

and a protonated amino group. Initially, baclofen (R-enantiomer) was manually docked in the

closed form model of GABAB1 receptor, the carboxylate moiety closed to the side chain of

Ser246 and its γ-amino group facing the side chain of Asp471. In this position, the chlorophenyl

group is pointing toward lobe-II and is in the proximity of Tyr366. In order to suppress the steric

hindrances and geometric inconsistencies, the ligand-protein complex was submitted to

molecular mechanics calculations using the Discover 3.00 calculation engine with the CFF

forcefield (Insight II version 980, MSI). The non-bond cut-off method and the dielectric

constant were respectively set up as cell multipole and distance-dependent (ε= r). Initially,

energy minimization were performed using a Steepest Descent algorithm (until the maximum

derivative was less than 2kcal/mol/Å) followed by a Conjugated Gradient algorithm(until the

maximum derivative was less than 0.01kcal/mol/Å), while the Cα-trace was tethered with a

quadratic potential. Then molecular dynamics was applied to the minimized system at constant

volume and temperature (298 K). The integration time step was set up to 1fs. During the

dynamics, the force constant value of the quadratic potential was reduced from 100 to 60, 30, 20,

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10 and 0 every 40ps. A snapshot of the system was saved every 400fs. Once the system was

equilibrated, the coordinates of 20 snapshots were averaged and resubmitted to the previously

described minimization protocol with no Cα restraints. Contacts between the ligand and the

protein were subsequently analyzed using the web interface of the WHATIF program

(http://swift.embl-heidelberg.de/servers2/ ).

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RESULTS

Identification of putative structural regions of the GABAB1 receptor involved in ligand

binding - Several splice variants have been identified for the GABAB1 receptor which all

possess an identical ligand binding domain but differ in their extreme N-terminal sequence (24)

or in their heptahelix regions (36, 37). All our studies have been performed with the rat

GABAB1a receptor (the first Met being residue number 1). A 3D model of the GABAB1 binding domain

has been constructed based on the structure of the open forms of LBP and LIVBP (Fig. 1a).

According to this 3D model and the known structural elements involved in leucine binding in

LIVBP, and in amide binding in AmiC (see Fig. 1), the GABA binding site would be formed by:

a) the loop between strand βC and helix αIII (loop βC-αIII according to the nomenclature of the

secondary stuctural elements of LIVBP proposed by Sack et al. (18), residues 243-250), b) the

N-terminal portion of the loop βD-βE (residues 266-272), and c) the N-termini of helices αI

(residues 184-192) and αIX (residues 463-473) (Fig. 1). We therefore undertook a systematic

analysis, by site directed mutagenesis, of all residues that can interact with ligands within these

regions (see Fig. 1b). Each mutant was analyzed for its ability to bind [125I]-CGP64213, and to

be activated by the agonists GABA and baclofen (Fig. 2).

Occupancy of the GABAB1 subunit by GABA or baclofen plays a pivotal role in the

activation of the heteromeric receptor - We have previously reported that several mutations of

the GABAB1 subunit either increase or decrease the potency of GABA and baclofen is

displacing [125I]-CGP64213 bound to the GABAB1 subunit expressed alone (see Table 1). The

effect of these mutations, as well as additional ones generated during this study, on the potency

of GABA and baclofen at the heteromeric GABAB receptor was analyzed after co-expression of

the different GABAB1 mutants with the wild-type GABAB2 subunit in HEK293 cells. The

activation of the GABAB heteromeric receptor was analyzed by measuring the stimulation of

phospholipase C via the chimeric G-protein Gqi9 (32, 38). The potencies (EC50 values) of both

GABA and baclofen were measured on the different heteromers and compared to the binding

affinity (IC50 values which, under our experimental conditions, are similar to Ki) of both GABA

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and baclofen as determined by displacement of bound [125I]-CGP64213 (see Table 1) on

GABAB1 mutants expressed alone. A good correlation was found between GABA (Fig. 3) and

baclofen (data not shown) binding affinity values (IC50) on most GABAB1 mutants and the

EC50 values measured with our functional assay on the mutant heteromeric receptors. These data

show that agonist binding at the GABAB1 receptor is critical for the activation of the

heteromeric GABAB receptor and validates the use of such a functional assay to further analyze

the molecular determinants of agonist binding at the GABAB1 receptor for which no [125I]-

CGP64213 binding could be measured.

Ser246 of GABAB1a receptor is critical for agonist binding and activity - The mutation of a

residue interacting with GABA in the GABAB1 subunit is expected: 1) to either largely decrease

the potency of GABA in displacing [125I]-CGP64213 binding or to suppress [125I]-

CGP64213 binding and 2) to largely decrease the potency of agonists in activating the receptor.

Within the loops βC-αIII and βD-βE, the mutation of Ser246, Ser265 and Tyr266 were found to

largely decrease [125I]-CGP64213 binding (Fig. 4), but only the mutation of Ser246 affected

the potency of GABA and baclofen at the GABAB receptor (Fig. 1b, Table I). As shown in Fig.

4, [125I]-CGP64213 did not bind to the S246A mutant although correctly expressed in HEK

293 cells. Both GABA and baclofen were able to activate the heteromeric receptor containing the

S246A mutated GABAB1 subunit, indicating a correct expression, folding and dimerization of

the receptor. However, the EC50 values of both agonists were increased by a factor 1000

compared to those determined with the wild-type receptor (Fig. 5). Interestingly, Ser246 aligns

with Ser79 of LIVBP which interacts with ligands (Fig. 1b) (18) thus suggesting that the

hydroxyl group of Ser246 directly interacts with the agonists GABA and baclofen. In agreement

with this proposal, mutation of this residue into the more bulky Asn residue, or into Pro which

may affect the topology of this loop, suppressed both [125I]-CGP64213 binding and agonist

activation of the receptor (Table I and Fig. 5). Furthermore, even the replacement of Ser246 by a

Thr residue resulted in a large decrease in agonist potency (Table I and Fig. 5), suggesting that

the orientation of the hydroxyl group is critical for a high agonist potency. All these latter

mutants were found to be correctly expressed and targeted to the plasma membrane when co-

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expressed with the wild-type GABAB2 subunit (data not shown).

Exploring helix I and IX: Asp471 is a critical residue for agonist action - We applied the

same strategy to identify residues possibly interacting with agonists within helices I and IX. The

mutation of Phe463, Tyr470 and Asp471 were found to largely decrease [125I]-CGP64213

binding (Fig. 4), but only the mutation of Asp471 affected the potency of GABA and baclofen to

activate the GABAB receptor (Fig. 1b, Table I). As shown in Fig. 4, no [125I]-CGP64213

binding could be measured on membranes expressing the Asp471 mutant receptor. Moreover,

co-expression of the mutant D471A with the wild-type GABAB2 receptor did not lead to a

functional response upon application of GABA or baclofen (Fig. 6a,b). Western blots clearly

show that the lack of function of this mutant receptor was not due to a lack of expression (Fig.

3b). Moreover, the mutant D471A was correctly inserted in the plasma membrane when co-

expressed with the GABAB2 subunit (Fig. 6c), as shown by the large amount of biotinylated

subunits after exposure of the intact cells to the non permeant protein reagent, sulfo-NHS-

Biotin. Taken together, these data reveal that Asp471 is a critical residue for agonist binding in

GABAB1 receptors. In agreement with this proposal, the conservative mutation of this Asp into

Glu was sufficient to dramatically reduce [125I]-CGP64213 binding. However, a significant

increase in IP formation could be detected upon application of either GABA or baclofen to cells

co-expressing D471E and the wild-type GABAB2 receptor, showing a very partial recovery of

the agonist action at this mutant receptor (Fig. 6). This partial recovery is likely to have been

underestimated since the expression level of the D471E mutant was lower than that of the wild-

type or D471A mutant (Fig. 4b).

Tyr366 in lobe-II decreased GABA and baclofen affinity and converts baclofen into an

antagonist - In PBPs, or related proteins, which have been crystallized in a closed form

complexed with a ligand , residues from both lobes contact the ligand (16, 19, 39-41). We

therefore examined the role of residues within lobe-II in ligand binding and agonist activity. We

focused our efforts on residues within the loops βF-αV and βH-αVII because they both face the

putative GABA binding site, and a residue interacting with amide in AmiC is located within one

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of these loops (Fig. 1a, 8a, c). None of the alanine substitutions generated in these loops prevent

[125I]-CGP64213 binding (Fig. 1b, Table I, Fig. 3a). The IC50 values of both GABA and

baclofen for displacement of [125I]-CGP64213 binding were similar to those of the wild-type

receptor for all these mutants except Y366A. For this mutant, the IC50 for values of these two

agonists were increased by a factor 50-100 (Table I and Fig. 7a) whereas displacement by cold

CGP64213 was not affected (1.1 ± 0.1 nM for the wild type, 1.0 ± 0.4 nM for Y366A, n=3).

Although GABA and baclofen displayed a similar affinity for this mutant (Fig. 7a), the Y366A

subunit formed a functional receptor activated by GABA but not by baclofen (Fig. 7b). In cells

co-expressing this receptor and GABAB2, GABA stimulated IP formation by a factor 5-10, as

observed with the wild-type receptor, indicating a correct expression, targeting to the plasma

membrane and association with the wild-type GABAB2 subunit. However, no significant

formation of IP could be measured on this mutant receptor with baclofen (Fig. 7b). Accordingly,

if GABA and baclofen bind to the same site in GABAB1, baclofen is expected to act as an

antagonist at this mutant receptor (or as a very partial agonist). Indeed, as shown in Fig. 7c, 5

mM baclofen was found to inhibit the action of GABA at this mutant receptor in a competitive

manner. Thus, the mutation Y366A converts baclofen into a competitive antagonist.

Molecular modeling of the GABAB1 extracellular domain in a closed form and docking of

baclofen - In order to rationalize the effect of the mutations described above, the position of the

mutated residues were visualized in the 3D model of the extracellular domain of the GABAB1

−receptor described above (Fig. 1a). As shown in Fig. 8a and 8c, Ser246 and Asp471 are correctly

located to interact with GABA in lobe-I. The distance between the hydroxyl and carboxylic

groups of these two residues (10 Å) is compatible with this possibility, however the side chain of

Tyr366 in loop βH-αVII of lobe-II, although facing the putative GABA binding pocket in lobe-

I, is located more than 15 Å from these residues. As such, this open form model could not easily

explain the binding and functional properties of the Y366A mutant.

We therefore undertook the construction of a 3D model for a closed form of the GABAB1

binding domain using the coordinates of a 3D model of the closed form of LIVBP (42) kindly

provided by F. Quiocho, and the coordinates of the closed liganded form of AmiC (16). In the

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closed form model, Tyr366 comes in close proximity to Ser246 and Asp471. Baclofen, which

corresponds to a chlorophenyl derivative of GABA (see Fig. 2) has been manually docked in this

3D model, with its carboxyl group facing Ser246, and its amino group facing Asp471. The model

has then been subjected to simulation protocols as described in the Materials and Methods

section. In this model, the carboxyl group of baclofen forms H-bonds with the hydroxyl group of

Ser246 (from lobe-I) and those of Tyr366 (from lobe-II) (distance O-O <3Å), and its amino

group forms a salt bridge with the carboxylate of Asp471 (distance N-O <3Å) (Fig. 8d). This

latter interaction is stabilized by the aromatic ring of Tyr266 and a H-bond with the backbone

(carbonyl of Pro467). The chlorophenyl group of baclofen was found to fit into a pocket lined by

Tyr366 and Trp394 from lobe-II, and Leu468 from lobe-I.

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DISCUSSION

Our mutagenesis and modeling study has identified a set of residues in the GABAB1a

subunit which are critical for agonists binding and activation of the heteromeric GABAB

receptor. Our data suggest that two residues in lobe-I, Ser246 and Asp471 and one residue in

lobe-II interact with GABA and baclofen. Interestingly, the mutation of Tyr366 into Ala, not

only decreases GABA and baclofen affinity but also converts baclofen into an antagonist.

We have previously reported that Ser246 is critical for [125I]-CGP64213 binding at

GABAB1a receptors (15). Using a functional assay, we show here that this residue plays a critical role in

both GABA and baclofen action, their apparent affinity being decreased by a factor 1000 when

Ser246 is mutated into Ala or even Thr. In accordance with to the possible importance of this

residue, a search within the Drosophila and C. elegans genomes for GABAB1-like receptors

shows that this residue is conserved through evolution (43). Moreover, this residue aligns with

Ser79 of LBP and LIVBP, the hydroxyl group of which forms H-bonds with the α-carboxylic

group of leucine (18) (Fig. 9). Ser246 also aligns with Ser85 of AmiC which interacts with

acetamide (Fig. 1) (16). This Ser residue is also conserved in all mGlu receptors and has been

proposed to form H-bonds with the α-carboxylic group of glutamate (Fig. 9) (13, 14).

Accordingly we propose that Ser246 of GABAB1a also contacts via a H-bond the carboxylic

group of GABA and baclofen. This proposal is consistent with both our modeling studies, and

the identification of Asp471 as possibly forming an ionic interaction with the amino group of

these GABAB agonists (see below).

In addition to this Ser residue, the hydroxyl group of a Thr102 in LIVBP (Fig. 1b) forms H-

bonds with the α-amino group of leucine (18). A conserved Thr has been proposed to play the

same role in mGlu receptors (13, 14) (Fig. 9). In GABAB1a, this Thr residue aligns with Ser269

which does not play a critical role in GABA and baclofen binding. Interestingly, neither GABA

nor baclofen possess an α amino acid moiety (Fig. 9). Indeed we previously reported that Ser269

was involved in the effect of Ca2+ on this receptor subtype (43).

Among all other mutated residues from lobe-I, only Asp471 was found to play a critical role

in the action of GABA and baclofen. Mutation of this residue into Ala suppresses antagonist

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binding as well as GABA and baclofen induced activation of the receptor. This total loss of

function did not result from a lack of expression of this mutant protein. Moreover, the D471A

mutant was found to be correctly inserted into the plasma membrane when co-expressed with

the GABAB2 receptor. Since GABAB2 is required for the plasma membrane localization of the

GABAB1 receptors (28, 44), these data also indicate that the mutation of Asp471 does not affect

the formation of the heterodimer. In agreement with such an important role of Asp471 in agonist

binding, this residue is conserved in Drosophila and C. elegans GABAB1-like receptors (data

not shown). Moreover, our modeling studies revealed that the position of this residue allows its

side chain to correctly interact with the amino group of GABA. Indeed, this residue is located at

the bottom of the binding pocket, and points toward a hydrophobic environment which lacks any

polar residue in LIVBP and constitutes the binding pocket for the side chain of leucine (18). In

this region in mGlu receptors, the side chain of an Arg (Arg78) points toward the binding pocket

(Fig. 9) and has been proposed to interact with the γ carboxylic group of glutamate in mGlu4

(14) and mGlu1 (45) receptors.

Taken together, our data show that leucine, glutamate and GABA interact in a similar

binding pocket in lobe-I of LIVBP, mGlu and GABAB1 receptors respectively (Fig. 9). They

also highlight how the same binding pocket in lobe-I has evolved within these LIVBP-like

proteins to specifically recognize different but related molecules (Fig. 9).

Our study also identified 4 residues within lobe-I, Ser265, Tyr266, Phe463 and Tyr470 the

mutation of which dramatically decrease [125I]-CGP64213 binding, but does not change the

properties and apparent affinity of both GABA and baclofen. Although the lack of significant

binding to the S265A, Y266F and Y470F mutants may result from their low expression level,

this cannot be the case for the F463A and Y470A mutants (data not shown and Fig. 4). One may

therefore propose that these two residues are required for the binding of this large GABAB

antagonist which possesses two aromatic moieties (Fig. 2). However, additional experiments are

required to better characterize the role of these residues in GABAB antagonists binding and

action.

The data discussed above demonstrate the pivotal role of lobe-I in ligand binding to the

GABAB1 receptor, and in the agonist activation of the heteromeric receptor. However, our data

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also revealed that the mutation of Tyr366 from lobe-II largely decreases the affinity of both

GABA and baclofen. This mutant subunit is correctly expressed and targeted to the plasma

membrane when associated with the wild-type GABAB2 subunit, as shown by its activation by

GABA which is to an extent similar to that obtained with the wild-type receptor. This large

decrease in affinity suggests therefore, that Tyr366 has either direct or indirect contact with these

agonists. Such an interaction does not appear to be possible in our 3D model for an open form of

the GABAB1 binding domain. However, in a closed form model, the hydroxyphenyl of Tyr366

points towards the GABA binding site in lobe-I, and its oxygen forms a H-bond with the

carboxylic group of baclofen. Further experiments are required to validate this possibility.

However, these data already show that residues in lobe-II affect the affinity of agonists, in

agreement with the observation that residues from both lobes contact the ligands in many PBP-

like proteins (16, 19, 39-41). Interestingly, this mutation also prevents baclofen from activating

the heteromeric receptor, and converts it into a competitive antagonist. In our 3D model, the

chlorophenyl group of baclofen points towards a pocket lined by the hydroxyphenyl group of

Tyr366. It is therefore possible that the replacement of this Tyr by an Ala changes the position

of surrounding residues such that the chlorophenyl group in no longer accepted in a closed-

active conformation of the binding domain, therefore converting baclofen into an antagonist.

Although additional work is required to demonstrate this hypothesis, it is interesting to note that

a mutation in lobe-II, lining the binding pocket of AmiC, converts the bulky butyramide from an

antagonist to an agonist (46).

The GABAB receptor requires the presence of two subunits, GABAB1 and GABAB2 for

its full activity. Although one role of GABAB2 is to allow the correct insertion of GABAB1 in

the plasma membrane (28, 44), other roles in receptor functioning have not yet been

characterized. Our data show a correlation between the affinity of both GABA and baclofen

measured in binding studies on wild-type or mutant GABAB1 receptors and their EC50 values

measured in a functional assay after co-expression with GABAB2 receptor. Moreover, the

mutation of Asp471, a residue that is likely to play a critical role in GABA binding according to

our modeling studies, suppresses the function of the heteromer. Finally, a single mutation in the

GABAB1 subunit is sufficient to convert the selective heteromeric GABAB receptor agonist

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baclofen into an antagonist. Taken together, and in agreement with recent additional data (47),

these results add further strength to the proposal that GABA binding on the GABAB1 subunit is

required and is the main determinant for the activation of the heteromer. Comparison of the

putative binding pocket of GABAB1 and GABAB2 reveals that although Asp471 of GABAB1

is conserved in GABAB2, Tyr366 and Ser246 are replaced by Asp and Pro in GABAB2

respectively (see Fig. 1b). The mutation of Ser246 into Pro in GABAB1 is sufficient to suppress

the action of GABA on the heteromer. However, GABA and baclofen have been shown to

occasionally activate the GABAB2 receptor expressed alone (25, 48), suggesting that the

GABAB2 receptor is able to bind these two agonists. Our data indicate that if GABA and baclofen bind

to the GABAB2 subunit, they have to do it in a different way from GABAB1.

In conclusion, our data strongly support the importance of lobe-I for binding properties of

agonists in family 3 heptahelix receptors. They also reveal that residues in lobe-II can be critical

for the agonist property of family 3 receptor ligands. This is consistent with the proposal that the

closure of the two lobes (the so-called Venus fly-trap mechanism of action (19)) constitutes a

key step in family 3 receptor activation.

Acknowledgements - The authors express their special thanks to Drs. M.-L.

Parmentier, F. Acher, G. Labesse and J. Bockaert for constant support and

constructive discussion all along this work. The authors wish to thank Mss. F.

Gaven and A. Boncompain for their help for some experiments. Drs. F. Quiocho

(Houston, Texas, U.S.A.) and P. O’Hara (ZymoGenetics Inc, Seattle, Washington,

U.S.A.) are acknowledged for the coordinates of the closed form model of LIVBP.

We thank Drs. B. Mouillac, T. Durroux, A. Dumuis, C. De Colle and F. Carroll

(CCIPE, Montpellier, France) for critical reading of the manuscript.

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FOOT NOTES

1 The abbreviations used are: AmiC, Regulatory subunit of the amidase operon from P.

aeruginosa; CaS, Ca2+-sensing; GABA, γ-amino-n-butyric acid; GABAB , γ-aminobutyric

acid type B; LBP, leucine-binding protein; LIVBP, leucine/isoleucine/valine-binding protein;

mGlu, metabotropic glutamate; PBPs, periplasmic binding proteins.

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FIGURE LEGENDS

Figure 1: Regions and residues of the GABAB1a receptor binding site selected for mutagenesis.

a) Ribbon view of a 3D model of the extracellular GABAB1 receptor domain constructed based

on the structure of the open form of LIVBP and LBP. The regions selected for mutagenesis are

shown in yellow. b) Sequence of the regions selected for mutagenesis in GABAB1 receptor

aligned with the corresponding regions of mGlu receptors, GABAB2 receptor, LIVBP, LBP and

the AmiC protein. Residues that interact with leucine in LIVBP, and with acetamide in AmiC,

and those proposed to interact with glutamate in mGlu receptors are highlighted in black.

Residues which have been mutated in GABAB1 receptor are in bold. Residues of GABAB1a

receptor identified are being critical for agonist binding (Ser246 and Asp471) and baclofen

activity (Tyr366) are highlighted in black. Residues underlined correspond to the number

indicated on top of the sequence.

Figure 2: Structure of the GABAB ligands used in this study.

Figure 3: The binding affinities of GABA on several GABAB1 receptor mutants correlates with

their EC50 values determined on the heteromeric receptor containing the GABAB1 receptor

mutant and the wild-type GABAB2 receptor. The GABA binding affinity (IC50 values not

different from Kd according to our experimental conditions) on the indicated GABAB1 receptor

mutants was determined by displacement of [125I]-CGP64213 binding. The EC50 value of

GABA in stimulating IP formation was determined using cells expressing the indicated

GABAB1 receptor mutant together with the wild-type GABAB2 receptor and the mutated G-protein

Gqi9. The correlation observed is highly significant. Values are means of several experiments

and were taken from table I. A similar correlation was also observed with baclofen (data not

shown).

Figure 4: Effect of some mutations on the specific binding of [125I]-CGP64213 (a) and on the

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expression level of the protein after transient expression in HEK 293 cells (b). In a) 10 µg of

membrane proteins were used and the specific binding of [125I]-CGP64213 was measured

using 1 nM of this radioligand. In b) 10 µg of membrane proteins were used and the GABAB1

receptor protein was detected using an antibody directed against its C-terminus. Results are from

one experiment representative of at least two others.

Figure 5: The mutation of Ser246 into either Ala, Pro or Asn dramatically affects GABA and

baclofen affinities. Typical dose effect of GABA (a) and baclofen (b) in stimulating IP formation

in cells expressing the indicated GABAB1 receptor mutant together with the wild-type

GABAB2 receptor and the mutated G-protein Gqi9. Values are means ± s.e.m. of triplicate determinations

taken from a typical experiment.

Figure 6: The mutation of Asp471 into either Ala or Glu inhibits agonist activities of both GABA

and baclofen. Typical dose effect of GABA (a) and baclofen (b) in stimulating IP formation in

cells expressing the indicated GABAB1 receptor mutant, the wild-type GABAB2 receptor and

the mutated G-protein Gqi9. In (c), the D471A mutant is shown to be correctly targeted to the

cell surface when co-expressed with the wild-type GABAB2 receptor. After biotinylation of the

cell surface proteins, the total amount GABAB1 receptor protein (lane 1) or the amount bound

(lane 3) or not (lane 2) to the streptavidin beads was estimated by western blotting. The

experiment was conducted with cells expressing GABAB1 receptor (BR1) alone or co-

expressed with GABAB2 receptor (BR2), the D471A mutant alone or co-expressed with

GABAB2 receptor. Note that a higher amount of GABAB1 receptor or D471A proteins were biotinylated

(and therefore located at the cell surface) when co-expressed with GABAB2 receptor.

Figure 7: The mutation of Tyr366 in GABAB1 receptor decreases GABA affinity and converts

baclofen into an antagonist. a) Displacement of [125I]-CGP64213 binding from the wild-type

GABAB1 receptor (open symbols) or from the Y366A mutant (closed symbols) with GABA

(circles) or baclofen (squares). b) Stimulation of IP production by GABA (circles) or baclofen

(squares) in cells expressing wild-type GABAB1 receptor (open symbols) or the Y366A mutant

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(closed symbols) with the GABAB2 receptor and the Gqi9 chimera. c) Effect of various

concentration of GABA applied alone (closed circles) or in the presence of 5 mM baclofen

(open circles) on the stimulation of IP production in cells expressing Y366A with GABAB2

receptor. Data are means ± s.e.m. of triplicate determinations from a typical experiment (each

repeated at least 3 times with similar results), and are expressed as percentage of the [125I]-

CGP64213 specific binding (a), or percentage of the maximal effect obtained with 10 mM

GABA (b, c).

Figure 8: Ribbon views of the 3D models of putative open (a) and closed (b) conformations of

the GABAB1 receptor binding domain (the insertion comprising residues 488 to 502 is not

shown in (a)). Close view of the cleft that separate the two lobes in the open (c) and closed (d)

conformation. In (c) and (d) the residues identified as being important for GABA and baclofen

action (Ser246, Asp471 and Tyr366) are shown. Baclofen docked on the closed conformation is

shown in panel d. A closer view of baclofen in its binding pocket in the model of the closed

conformation of the GABAB1 receptor, showing the ionic interaction between the amino group

of baclofen with the side chain of Asp471, and the H-bonds of the carboxylic group of baclofen

with the side chain of Ser246.

Figure 9: Differences and similarities in the ligand binding pocket of LIVBP, GABAB1 receptor

and mGlu1 receptor.

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Table I: Effect of various mutations in GABAB1 receptor on the affinity of both GABA and

baclofen as determined by displacement of [125I]-CGP64213 binding (IC50 in µM) and receptor

activation (EC50 in µM).

mutation GABA baclofen CGP binding IP assay CGP binding IP assay IC50 (µM) EC50 (µM) IC50 (µM) EC50 (µM)

WT 22.3 ± 1.9 2.9 ± 0.6 30 ± 2 5.0 ± 1.3

lobe-I

S246A N.B. a >1000 N.B. >1000 S246P N.B. N.E. N.B. N.E. S246T N.B. >1000 N.B. - S246N N.B. N.E. N.B. N.E.

loops S247A 5.32 ± 0.88 a 0.37 ± 0.09 b 11.3 ± 0.9 b 0.38 ± 0.04 bβC-αIII S249A 26.2 ± 13.3 a - - -

& T250A 36.4 ± 12.8 a - - -βD-βE S265A N.B. 1.9±0.4 N.B. -

Y266F N.B. 3.9 ± 1.3 N.B. 18.3 ± 5.1 S268A 27.8 ± 3.4 a 1.02 ± 0.15 b 43.0 ± 4.1 b 0.91 ± 0.41 b S269A 238 ± 63 a 14.9 ± 0.8 b 28.8 ± 5.4 b 1.78 ± 0.22 b S270A 406 ± 26 a 24.5 ± 8.1 332 ± 1 51 ± 11

C187A 36.8 ± 3.6 a 1.8±0.3 - - E192A 22.3 ± 6.6 - - - E458A 16.5 ± 0.8 a - - - E459A 35.0 ± 0.1 a - - -

helices T460A 42.1 ± 4.7 a - - -αI & αIX F463A N.B. 3.23 ± 0.67 N.B. 12.6 ± 1.0

Q464A 29.3 ± 7.6 a - - - E465A 63 ± 28 114 ± 30 - 78.0 ± 5.5 Y470A N.B. a 1.74 ± 0.69 N.B. - Y470F N.B. 0.96 ± 0.47 N.B. - D471A N.B. N.E. N.B. N.E. D471E N.B. N.E. N.B. N.E.

lobe-II

Q312A 4.1 ± 0.9 a 0.51 ± 0.21 8.0 ± 1.4 a 1.04 ± 0.16loop Q313A 13.4 ± 0.8 a - - -

βF-αVI T314A 9.9 ± 3.6 a - - -

T315A 43.6 ± 1.5 a - - - E316A 61.1 ± 17.3 a - - -

loop F365A 17.6 ± 0.1 8.2± 6.1 30.8±4.5 2.6±0.7βH-αVII Y366A 3420 ± 522 97.1 ± 5.2 1559 ± 28 ANTAGO

E367A 35.4 ± 8.2 6.35 ± 4.25 14.9±3.2 28.0±0.6

Mutants highlighted in bold are those we proposed to be critical for agonist binding and action.

N.B.: specific [125I]-CGP64213 is less than 10% of that measured on membranes expressing

the wild-type receptor. N.E.: no significant stimulation of IP formation with 1 mM of the

agonist. a and b: values are taken from (15) and (43), respectively.

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Jean-Philippe R. PinWolfgang Froestl, Bernhard Bettler, Hugues-Olivier Bertrand, Jaroslav Blahos and Thierry C. Galvez, Laurent Prezeau, Gerald Milioti, Miloslav Franek, Cecile Joly,

activation process of GABAB receptorsMapping the agonist binding site of GABAB Type 1 subunit sheds light on the

published online September 13, 2000J. Biol. Chem.

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http://www.jbc.org/lookup/doi/10.1074/jbc.M007848200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M007848200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/09/13/jbc.M007848200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2000/09/13/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/09/13/jbc.M007848200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

mapping the agonist binding site of gabab type 1 subunit sheds

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