COMMUNICATION
Identification of a GABAB Receptor Subunit, gb2,
Required for Functional GABAB Receptor Activity*
Gordon Y. K.
Ngabc,
Janet
Clarkbde,
Nathalie
Coulombeab,
Nathalie
Ethierf,
Terence E.
Hebertf,
Richard
Sullivana,
Stacia
Kargmana,
Anne
Chateauneufa,
Naohiro
Tsukamotog,
Terry
McDonaldh,
Paul
Whitingi,
Éva
Mezeyj,
Michael P.
Johnsonh,
Qingyun
Liuh,
Lee F.
Kolakowski Jr.k,
Jilly F.
Evansh,
Tom I.
Bonnerd, and
Gary P.
O'Neilla
From a Merck Frosst Center for Therapeutic Research,
Kirkland, Quebec H9H 3L1, Canada, the i Merck Sharp & Dohme
Research Laboratories, Terlings Park, Harlow, Essex CM20 2QR, United
Kingdom, g Banyu Pharmaceutical Co., Ltd., Tsukuba-shi,
Ibaraki-ken 300-2611, Japan, h Merck & Co., Inc., West Point,
Pennsylvania, 19486, f Montreal Heart Institute, Montreal,
Quebec H1T 1C8, Canada, d National Institutes of Mental Health,
Section on Genetics and j NINDS, National Institutes of Health,
Bethesda, Maryland 20892-4094, and the k Departments of
Pharmacology and Biochemistry, University of Texas Health Science
Center at San Antonio, San Antonio, Texas 78284
 |
ABSTRACT |
G protein-coupled receptors are
commonly thought to bind their cognate ligands and elicit functional
responses primarily as monomeric receptors. In studying the recombinant
-aminobutyric acid, type B (GABAB) receptor (gb1a)
and a GABAB-like orphan receptor (gb2), we observed that
both receptors are functionally inactive when expressed individually in
multiple heterologous systems. Characterization of the tissue
distribution of each of the receptors by in situ
hybridization histochemistry in rat brain revealed co-localization of
gb1 and gb2 transcripts in many brain regions, suggesting the
hypothesis that gb1 and gb2 may interact in vivo. In three
established functional systems (inwardly rectifying K+
channel currents in Xenopus oocytes, melanophore pigment
aggregation, and direct cAMP measurements in HEK-293 cells), GABA
mediated a functional response in cells coexpressing gb1a and gb2 but
not in cells expressing either receptor individually. This GABA
activity could be blocked with the GABAB receptor
antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2
receptors, co-immunoprecipitation of gb1a and gb2 receptors was
demonstrated, indicating that gb1a and gb2 act as subunits in the
formation of a functional GABAB receptor.
 |
INTRODUCTION |
Metabotropic
GABAB1 receptors
were first distinguished pharmacologically by Hill and Bowery (1).
Kaupmann et al. (2) recently cloned two alternatively
spliced forms of the GABAB receptor, termed gb1a and gb1b,
which belong to the G protein-coupled receptor superfamily and are most
closely related to the metabotropic glutamate receptors. Although
native GABAB receptors are reported to activate inwardly
rectifying K+ channels (Kir) (3), recombinant gb1a
receptors coexpressed with Kir channels in Xenopus oocytes
failed to be functionally active (2, 4, 12). A recent report has shown
that recombinant GABAB receptors fail to couple to effector
pathways in a variety of non-neuronal and neuronal cell types,
suggesting that additional cellular component(s) are required (4).
Failure to show GABAB receptor function coupled with
previous reports that GPCRs can undergo dimerization (5-8), suggested
that heterodimerization may be important for GABAB receptor
function. Furthermore, receptor heterodimerization appears to rescue
function of mutated or chimeric muscarinic and adrenergic receptors
(9). We report here that coexpression of gb1 and gb2 receptors are
necessary for the formation of functional GABAB receptors
and result in heterodimerization.
 |
EXPERIMENTAL PROCEDURES |
Expression Constructs for gb1a and gb2 Receptors--
The murine
gb1a (mgb1a) cDNA was constructed from two expressed sequence
tags (IMAGE Consortium clone identification numbers 472408 and 319196), combined by standard PCR methods, and subcloned into the
pCINeo (Stratagene) and pcDNA3.1 (Invitrogen)
vectors.2 The rat gb1a
receptor was obtained by PCR of rat brain cDNA using oligonucleotide primers based on the published sequence (Ref. 2;
GenBankTM accession number Y10369) and subcloned into
pcDNA3.1.
Two independently derived cDNAs of human gb2 (GenBankTM
accession numbers AF069755 and AF056085, the former having previously been called GPR51 (10)) were used to make expression constructs. A
N-terminal FLAG-tagged hgb2/pcDNA3.1 construct encoding a modified influenza hemaglutinin signal sequence (MKTIIALSYIFCLVFA) followed by
an antigenic FLAG (DYKDDDDK) epitope was generated by PCR (10). The
hgb2 construct used for expression in 293 cells was the coding sequence
from GenBankTM accession number AF056085 with the
C-terminal splice variant of GenBankTM accession number
AF0957233 inserted into
pcDNA3.1 in a manner similar to that for the rat gb1a.
In Situ Hybridization Histochemistry--
Adjacent coronal rat
brain sections were hybridized with labeled antisense and sense
riboprobes directed against rgb2 (GenBankTM accession
number AF058795) or rgb1 as described previously (11).4 Probes were generated
by amplification of rgb2 with JC216 (T3 promoter followed by bases
1172-1191) and JC217 (T7 promoter and the complement of bases
1609-1626) or with JC218 (T3 promoter and bases 2386-2405) and JC219
(T7 promoter and the complement bases 2776-2793) or by amplification
of rgb1a with JC160 (T3 promoter and bases 631-648) and JC161 (T7
promoter and the complement of bases 1024-1041). For colocalization
experiments, probes were either labeled with digoxigenin (rgb1a) or
35S (rgb2). Detection of the radiolabeled rgb2 probe was
performed using emulsion after detection of the digoxigenin-labeled
rgb1 probe on the same brain slices.
Melanophore Functional Assay--
Growth of Xenopus
laevis melanophores and fibroblasts and DNA transfections by
electroporation were performed as described previously (13). To monitor
the efficiency of transfection an internal control GPCR was used
(pcDNA1-cannabinoid 2). For Gi-coupled responses
(pigment aggregation), cells were preincubated in the presence of 100 µl/well of 70% L-15 medium containing 2.5% fibroblast-conditioned growth medium, 2 mM glutamine, 100 µg/ml streptomycin,
100 units/ml penicillin, and 15 mM HEPES, pH 7.3, for 30 min to induce pigment dispersion. Absorbance readings at 600 nm were
measured using a Bio-Tek Elx800 Microplate reader (ESBE Scientific)
before and after 1.5 h incubation with the ligands.
Stable and Transient Transfections and Determination of Cellular
cAMP Response in HEK-293 Cells--
Hgb2 and rgb1a cDNAs in
pcDNA3.1 were used to transfect HEK-293 cells. Stably expressing
cells were identified after selection in geneticin (0.375 mg/ml) by dot
blot analysis. For coexpression experiments the stable cell lines
hgb2-42 and rgb1a-50 were transiently transfected with hgb2 or rgb1a,
and cells were assayed for cAMP responses. Wild-type HEK-293 cells or
HEK-293 cells stably and transiently expressing the hgb2 and rgb1a
receptors were lifted in 1× phosphate-buffered saline, 2.5 mM EDTA, counted, pelleted, and resuspended at 1.5 × 105 cells/100 µl in Krebs-Ringer-HEPES medium (14), 100 µM Ro 20-1724 (RBI) and assayed for agonist-mediated
inhibition of forskolin-stimulated cAMP synthesis using a modified
version of an assay previously described by Kaupmann et al.
(2). cAMP determinations were made using a solid phase modification
(15) of the cAMP radioimmunoassay described by Brooker et
al. (16) and previously reported (17).
Kir Channel Activity--
Xenopusoocytes were isolated as
described (18). cDNA constructs for human Kir 3.1, Kir 3.2 channel
isoforms (gifts from Dr. Hubert Van Tol, University of Toronto),
Gi
1 (a generous gift of Dr. Maureen Linder, Washington
University), and cDNAs of mgb1a or FLAG-hgb2 (subcloned into pT7TS,
a gift of Dr. Paul Krieg, University of Texas) were linearized and
transcribed using T7 RNA polymerase and the mMessage mMachine (Ambion).
Individual oocytes were injected with 5-10 ng (in 25-50 nl) of Kir3.1
and Kir3.2 constructs with mRNAs for mgb1a or FLAG-hgb2 receptors with and without Gi
1. Recordings were made after 48 h as described (18). Standard recording solution was KD-98, 98 mM KCl, 1 mM MgCl2, 5 mM K-HEPES, pH 7.5, unless otherwise stated. Data
collection and analysis were performed using pCLAMP v6.0 (Axon
Instruments) and Origin v4.0 (MicroCal) software. For subtraction of
endogenous and leak currents, records were obtained in ND-96, 96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 5 mM Na-HEPES, and these were subtracted from recordings in KD-98 before further analysis.
Immunoprecipitation and Immunoblotting of GABAB
Receptors--
COS-7 cells (ATCC) were cultured and transiently
transfected with FLAG-hgb2 and mgb1a receptor DNAs alone and in
combination using LipofectAMINE reagent (Life Technologies, Inc.)
according to the manufacturer's instructions. Membranes prepared from
these cells were digitonin-solubilized, and mgb1a/FLAG-gb2 heterodimers were immunoprecipitated with either a mouse anti-FLAG M2 antibody (Kodak IBI) targeting the FLAG-gb2 receptor or anti-gb1 receptor rabbit
polyclonal antibodies 1713.1-1713.22 using previously
described conditions (6). The immunoprecipitates were then washed and
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted
with either the anti-FLAG or anti-gb1 antibodies using previously
reported conditions (6) and as described below.
 |
RESULTS AND DISCUSSION |
Tissue Distribution of gb1 and gb2 Receptor mRNAs--
Northern blot analysis revealed that many brain regions, including
cortex, show overlapping expression patterns for gb2 and gb1 receptor
mRNA (10). In situ hybridization in adjacent coronal sections of rat parietal cortex using 35S-labeled antisense
riboprobes indicates that mRNAs for both gb1 and gb2 receptors are
coexpressed in this brain region (Fig. 1, A and B). No hybridization signal was detected
with the control 35S-labeled sense gb1 and gb2 riboprobes
(data not shown). To examine expression at the cellular level,
digoxigenin-labeled gb1 and radiolabeled gb2 riboprobes were hybridized
to the same sections (Fig. 1, C and D). Overlay
of the gb1 and gb2 hybridization signals revealed that messages for
both receptors are generally expressed in the same neurons (Fig.
1E). In other major brain regions, including the
hippocampus, thalamus, and cerebellum,3 gb2 and gb1
receptor mRNAs are colocalized with
95% of gb2 expressing cells
also expressing gb1. This suggests that their coexpression may be
required for activity in these neurons.

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Fig. 1.
Colocalization of GABAB receptor
mRNAs by in situ hybridization histochemistry in
rat parietal cortex. Adjacent coronal sections of rat brain
showing parietal cortex hybridized with radiolabeled rgb1a
(A) and rgb2 (B) probes. Rat gb1 and gb2 probes
were labeled using 35S-UTP (A, B, and
D), and autoradiograms were developed after 4 weeks. For
colocalization studies the rat gb1 probe was digoxigenin-labeled and
developed using anti-digoxigenin horseradish peroxidase, the tyramide
signal amplification method and biotinyl tyramide followed by
streptavidin-conjugated CY3 (C). Red (CY3
positive) cells in C express gb1 receptor mRNA.
D shows autoradiography of the same field as in C
denoting hybridization to gb2 receptor mRNA. E is an
overlay of images C and D. Arrows
denote some of the double-labeled cells. Scale bar in
A and B, 2.0 mm; scale bar in
C-E, 50 µm.
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|
Recombinant gb1a Receptors Require Coexpression of gb2 for
Functional Activity--
In melanophores transiently cotransfected
with the mgb1a and hgb2 receptors, GABA mediated a
dose-dependent pigment aggregation response with an
IC50 value of 3-7 µM (n = 3), which is
absent in mock-transfected cells and cells transfected with the mgb1a or hgb2 alone (Fig. 2). The GABA-mediated
inhibitory activity represented 42-56% (n = 3) of a
control Gi-coupled CB2 cannabinoid receptor response (Fig.
2, inset). GABA activity could be inhibited by the CGP71872
antagonist (n = 3), indicating that this was
GABAB receptor-specific (Fig. 2).

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Fig. 2.
Functional complementation following
coexpression of gb1a and gb2 receptors in Xenopus
melanophores. GABA mediated a dose-dependent
aggregation response in melanophores coexpressing mgb1a and FLAG-gb2
( ) that could be blocked with 100 nM ( ) and 1 µM
CGP71872 ( ). The response of GABA on mock-transfected cells is shown
( ) as well as a control cannabinoid receptor subtype 2 response to
HU210 ligand (inset). This experiment is representative of
n = 4.
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|
To confirm the observations that functional GABAB receptor
results from the coexpression of gb1a and gb2 receptors and that it is
Gi-coupled, we directly examined modulation of cAMP levels in HEK-293 cells. In cell lines stably expressing the individual receptors we observed small and inconsistent responses in assays to
examine agonist-mediated modulation of cAMP synthesis (Fig. 3). However, transient hgb2 transfection
of HEK-293 cells stably expressing rgb1a (rgb1a-50) and transient rgb1a
transfection of HEK-293 cells stably expressing hgb2 (hgb2-42)
significantly enhanced the ability of baclofen and GABA to inhibit
forskolin-stimulated cAMP synthesis. Hgb2-42/rgb1a and rgb1a-50/hgb2
cells exhibited 28 and 34% reductions in forskolin-stimulated cAMP
synthesis with 30 µM baclofen and 40 and 43% decreases
with 30 µM GABA, respectively (Fig. 3). Although
inhibition of cAMP synthesis was sometimes observed with rgb1a-50/rgb1a
and hgb2-42/hgb2, these effects were relatively small (0-20%
inhibition; Fig. 3) when compared with those observed with the
coexpressing cells. Neither baclofen nor GABA in the absence of
forskolin had any effect on cAMP synthesis (Fig. 3). In addition,
wild-type HEK-293 cells did not exhibit baclofen- or GABA-mediated
inhibition of forskolin-stimulated cAMP synthesis (Fig. 3). These data
demonstrate that the functional GABAB receptor requires
both the gb1 and gb2 receptors for signaling via adenylyl cyclase.

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Fig. 3.
GABAB receptor modulation of
forskolin-stimulated cAMP synthesis in HEK-293 cells. HEK-293
cells stably expressing the hgb2 receptor (hgb2-42) or the rgb1a
receptor (rgb1a-50) were transiently transfected with hgb2 or rgb1a
expression plasmids to examine the effect of receptor coexpression on
modulation of cAMP synthesis. All transfected cells were tested with
300 µM baclofen or GABA (with 100 µM AOAA,
a GABA transaminase inhibitor, and 100 µM nipecotic acid,
a GABA uptake inhibitor) in the absence of forskolin and 30 µM baclofen or GABA in the presence of 10 µM forskolin. Wild-type HEK-293 cells were tested with
250 µM baclofen or 250 µM GABA in the
presence of 10 µM forskolin. Data are presented as the
percentage of total cAMP synthesized in the presence of forskolin only.
The data presented are from single representative experiments that have
been replicated twice. Fsk, forskolin; B,
baclofen; G, GABA with aminooxyacetic acid and nipecotic
acid.
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Native functional GABAB receptors have been reported to
couple to Kirs (3). Co-expression of the mgb1a and hgb2 with Kir 3.1/3.2 resulted in a significant stimulation of Kir current in response to GABA (301 ± 20.6% (n = 3) increase
over control current) measured at
80 mV, which could subsequently be
washed out with control solution (Fig. 4)
or blocked with the CGP71872 antagonist (data not shown).
Modulation of Kir 3.1/3.2 was not seen in oocytes expressing mgb1a or
hgb2 individually even in the presence of Gi
1 (Fig. 4).
The dependence of functional GABAB receptor activity on the
coexpression of gb1a and gb2 suggests that these receptors may undergo
heterodimerization.

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Fig. 4.
Coexpression of gb1a and gb2 permits GIRK
activation in Xenopus oocytes. A,
representative current families of Kir 3.1/3.2. Currents were evoked by
500-ms voltage commands from a holding potential of 10 mV, delivered
in 20 mV increments from 140 to 60 mV. B, in a protocol
designed to measure the effects of various receptors on Kir currents,
oocytes were held at 80 mV (a potential where significant inward
current is measured). Expression of mgb1a or FLAG-gb2 receptors alone
with or without Gi 1 resulted in no modulation of current
after GABA treatment. Co-expression of mgb1a and FLAG-hgb2 receptors
followed by treatment with 100 µM GABA resulted in
stimulation of Kir 3.1/3.2. Shown are representative traces from at
least three independent experiments under each condition.
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gb1a and gb2 Form a Heterodimer--
Immunoblot analysis revealed
selective expression of mgb1a monomers and homodimers in mgb1a and
mgb1a/FLAG-gb2 expressing cells and expression of FLAG-gb2 receptors in
FLAG-gb2 and mgb1a/FLAG-gb2 expressing cells (Fig.
5, lanes 1-8). To demonstrate
the existence of gb1a-gb2 heterodimers, we utilized a differential
co-immunoprecipitation and immunoblotting strategy. Anti-gb1 receptor
antibodies were used to blot receptors immunoprecipitated with
anti-FLAG antibodies (Fig. 5, lanes 9-12). No mgb1a
immunoreactivity was detected in samples prepared from mock vector
transfected cells, FLAG-gb2 expressing cells, and mgb1a receptor
expressing cells as expected because these species could not be
immunoprecipitated with the anti-FLAG antibody and detected with the
anti-gb1 antibody (Fig. 5, lanes 9-11). Immunoreactive
~250- (representing the mgb1a-gb2 heterodimer) and ~130-kDa species
(representing the mgb1a monomer) were detected only in cells
coexpressing the mgb1a and FLAG-gb2 receptors, demonstrating that gb1a
and gb2 can only be co-immunoprecipitated as part of a complex (Fig. 5,
lane 12). Similar demonstration of gb1a-gb2
heterodimerization was obtained when coexpressed receptors were
immunoprecipitated first with anti-gb1 antibodies followed by
immunoblotting with the anti-FLAG antibody (Fig. 5, lane
16). The ~250-kDa species represents the heterodimer, whereas
the ~130-kDa species represents the FLAG-gb2 monomer. No FLAG-gb2
immunoreactivity was detected in samples prepared from mock vector
transfected cells, FLAG-gb2 expressing cells and mgb1a receptor
expressing cells as expected because these species could not be
immunoprecipitated with the anti-gb1 antibody and detected with the
anti-FLAG antibody (Fig. 5, lanes 13-15). The gb1a-gb2
heterodimer, which is stable in SDS, might result from SDS-resistant
intermolecular transmembrane interactions as reported for the formation
of
2-adrenergic and dopamine D2 receptor homodimers (5, 6). The
monomer presumably results from partial disruption of protein-protein
binding domains (Sushi Repeats) or C-terminal
-helical domains in
mgb1a.2 Disulfide bonds may also contribute to dimer
formation as has been reported for the structurally related
metabotropic glutamate receptor (7).

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Fig. 5.
Co-immunoprecipitation of the murine gb1a and
FLAG-gb2 receptors and immunoblotting using reciprocal receptor
antibodies. Mgb1a and FLAG-gb2 receptors were expressed
individually or coexpressed in COS-7 cells. Immunoblots of the
solubilized membranes using gb1 receptor antibodies 1713.1-1713.2 show
selective expression of mgb1a receptors in mgb1a expressing cells
(lane 3) and mgb1/FLAG-gb2 coexpressing cells (lane
4) but not in mock transfected and FLAG-gb2 receptor expressing
cells (lanes 1 and 2). Immunoblot of the
solubilized membranes using the anti-FLAG-gb2 antibody shows selective
expression of FLAG-gb2 receptors in FLAG-gb2 expressing cells
(lane 6) and mgb1a/FLAG-gb2 coexpressing cells (lane
8) but not in mock transfected and mgb1a receptor expressing cells
(lanes 5 and 7). gb1a-gb2 heterodimers are
observed only in mgb1a/FLAG-gb2 coexpressing cells due to the fact that
the gb1 receptor was co-immunoprecipitated with the FLAG-gb2 receptor
using the FLAG antibody and detected with gb1 antibodies (lane
12). gb1 receptors are not detected in mock-transfected cells and
cells expressing gb1a or FLAG-gb2 (lanes 9-11). gb1-gb2
heterodimers are observed only in mgb1a/FLAG-gb2 expressing cells due
to the fact that the FLAG-gb2 receptor was co-immunoprecipitated using
the gb1 receptor antibodies and detected with FLAG antibody (lane
16). No FLAG-gb2 receptors are detected in mock-transfected cells
or cells expressing mgb1a or FLAG-gb2 (lanes 13-15). The
immunoblots shown are from one to three independent experiments.
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|
We have shown in three disparate expression systems that coexpression
of the gb1 and gb2 receptors is required for functional GABAB receptor activity. That the two receptors are
co-immunoprecipitated is consistent with the formation of heterodimers.
We cannot rule out the possibility that either receptor may function as
a monomeric or homodimeric receptor in the appropriate native cells.
Indeed there are regions of the brain, such as caudate/putamen, where gb1 mRNA is abundant and gb2 mRNA is undetectable (Fig. 1,
A and B). In such regions gb1 would either
require a different subunit or function as a monomer or homodimer.
However, our in situ hybridization analysis indicates that
in brain regions expressing gb2, gb1 is expressed in a majority of the
same neurons, suggesting that native receptors are heterodimers in
these cells. GPCRs are commonly thought of as monomeric receptors.
However, in light of our functional data and in situ
hybridization findings, it seems appropriate to consider the gb1 and
gb2 receptor proteins to be subunits of a functional GABAB receptor.
 |
Addendum |
During the editorial review of this manuscript,
similar work from four independent groups was published reporting the
formation of functional GABAB receptor heterodimers
(19-22).
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
b
These authors contributed equally to this work.
c
To whom correspondence should be addressed. Tel.:
514-428-3589; Fax: 514-428-4900; E-mail: gordon_ng{at}merck.com.
e
Supported by a Young Investigator Award from the National
Alliance for Research on Schizophrenia and Depression.
2
R. Sullivan, F. L. Kolakowski, Jr., M. P. Johnson, A. Chateauneuf, G. P. O'Neill, and G. Y. K. Ng, manuscript in preparation.
3
J. Clark, É. Mezey, A. S. Lam, and
T. I. Bonner, manuscript in preparation.
4
http://intramural.nimh.nih.gov/lcmr/snge/Protocol.html.
 |
ABBREVIATIONS |
The abbreviations used are:
GABA,
-aminobutyric acid;
GPCR, G protein-coupled receptor;
Kir, inwardly
rectifying K+ channel;
PCR, polymerase chain
reaction.
 |
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