Activation of nematode G protein GOA-1 by the human
muscarinic acetylcholine receptor M
2
subtype
Functional coupling of G-protein-coupled receptor and G protein
originated from evolutionarily distant animals
Masaomi Minaba
1
, Susumu Ichiyama
2
, Katsura Kojima
3
, Mamiko Ozaki
4
and Yusuke Kato
1
1 Immune Defense Unit, National Institute of Agrobiological Sciences, Ibaraki, Japan
2 Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Tokyo, Japan
3 Silk-Materials Research Unit, National Institute of Agrobiological Sciences, Ibaraki, Japan
4 Department of Biology, Faculty of Science, Kobe University, Japan
G-protein-coupled receptors (GPCRs) are membrane
receptors that are activated by specific agonist binding.
Activated GPCRs affect intracellular heterotrimeric
G proteins, which activate specific effectors (adenylyl
cyclase, phospholipase C, etc.) [1]. The heterotrimeric
G protein consists of Ga,Gb and Gc subunits [2,3].
The coupling specificity with GPCRs and effectors is
mainly determined by Ga, although Gb and Gc also
affect the specificity. Approximately 950 GPCR genes
have been found in the human genome, but only
17 Ga have been identified, which indicates that a sin-

October 2006, accepted 17 October 2006)
doi:10.1111/j.1742-4658.2006.05542.x
Signal transduction mediated by heterotrimeric G proteins regulates a wide
variety of physiological functions. We are interested in the manipulation of
G-protein-mediating signal transduction using G-protein-coupled receptors,
which are derived from evolutionarily distant organisms and recognize unique
ligands. As a model, we tested the functionally coupling GOA-1, Ga
i ⁄ o
ortho-
log in the nematode Caenorhabditis elegans, with the human muscarinic
acetylcholine receptor M
2
subtype (M
2
), which is one of the mammalian
Ga
i ⁄ o
-coupled receptors. GOA-1 and M
2
were prepared as a fusion protein
using a baculovirus expression system. The affinity of the fusion protein for
GDP was decreased by addition of a muscarinic agonist, carbamylcholine
and the guanosine 5¢-[3-O-thio]triphosphate ([
35
S]GTPcS) binding was
increased with an increase in the carbamylcholine concentrations in a dose-
dependent manner. These effects evoked by carbamylcholine were completely
abolished by a full antagonist, atropine. In addition, the affinity for carbamyl-
choline decreased under the presence of GTP as reported for M
2

q ⁄ 11
and Ga
12
)
[10]. Ga
o
is a member of the Ga
i ⁄ o
class. Caenorhabditis
elegans, a nematode, is a genetic model organism widely
used in laboratories [11]. The whole genome sequence of
C. elegans has been determined in multicellular organ-
isms [12]. In the genome of C. elegans,21Ga have been
found [13,14]. Although some Ga appear to be unique
in nematodes, orthologs of mammalian Ga
s
,Ga
q
,Ga
12
and Ga
i ⁄ o
have also been identified. GOA-1 is the Ga
i ⁄ o
ortholog in C. elegans [15]. GOA-1 is specifically
expressed in neurons in adults [16,17]. Knockout and
overexpression of goa-1, the gene encoding GOA-1,
affect some behaviors such as locomotion, egg-laying
and mating [16,17]. In addition, GOA-1 also regulates
the susceptibility to volatile anesthetic [18] and olfactory

::Ga
i1
fusion protein [25]. A large-
scale preparation of the fusion protein has been estab-
lished using a baculovirus expression system [25].
Therefore, M
2
receptor is one of the best models for
mammalian Ga
i ⁄ o
-coupled receptors. The effector regu-
lated by GOA-1 is still unclear, suggesting that GOA-1
activation should be directly measured to evaluate
the coupling of M
2
with GOA-1. We prepared an
M
2
mutant::GOA-1 fusion protein and directly assessed
the muscarinic-ligand-dependent activation of GOA-1.
Results
Expression of M
2
::GOA-1 fusion protein
The human M
2
receptor is a Ga
i ⁄ o
-coupled receptor.
To test whether M

(N-D)I3del::GOA-1. The membrane fraction of baculovirus-
infected cells was studied. The fusion protein was detected using
an alkaline phosphatase conjugated monoclonal antibody against
myc. The calculated mass for the recombinant protein (78 kDa) is
indicated by an arrow. Lane 1, wild-type virus infected cells; lane 2,
recombinant virus infected cells.
M. Minaba et al. Human GPCR activates nematode G protein
FEBS Journal 273 (2006) 5508–5516 ª 2006 The Authors Journal compilation ª 2006 FEBS 5509
In this recombinant protein, GOA-1 was directly fused
at the C-terminus of M
2
.Amyc-epitope tag was added
at the N-terminus of M
2
. To prevent rapid degrad-
ation, the central part of the third intracellular loop of
M
2
was deleted [26]. Asn at putative N-glycosylation
sites near the N-terminus of M
2
was mutated to Asp
to avoid diversified migration in western blot analyses
[27]. These modifications to M
2
do not interfere with
the M
2
–Ga
i ⁄ o

3
H]NMS) (Fig. 2A). This binding was
abolished by addition of the full antagonist, atropine.
In addition, the 50% inhibitory concentration (IC
50
)
for [
3
H]QNB displacement was estimated for atropine
(5 · 10
)8
m), and the full agonist, carbamylcholine
(5 · 10
)4
m) in the absence of GTP (Fig. 2B). These
IC
50
values are very like those with M
2
(N-D)I3del::
Ga
i1
(5.0 · 10
)8
m for atropine, and 3.3 · 10
)4
m for
carbamylcholine) [25]. These results indicate that M
2
in the GOA-1 fusion protein is functional, and the

m. The increase
of GTPcS binding was completely inhibited by
Fig. 2. Binding of muscarinic ligands. All
experiments were performed in triplicate.
Each data point represents the mean ± SEM.
(A) Binding of [
3
H]QNB and [
3
H]NMS to
myc-M
2
(N-D)I3del::GOA-1. The membrane
fraction containing 10 l g recombinant protein
was tested. (B) Displacement by atropine and
carbamylcholine of [
3
H]QNB binding. The
experiment was performed in the presence
(dotted line) or absence (solid line) of 1 m
M
GTP. [
3
H]QNB binding was normalized to the
value obtained at atropine ¼ 10
)13
M or
carbamylcholine ¼ 10
)7
M, respectively.

carbamylcholine of myc-M
2
(N-D)I3del::GOA-1 dec-
reased in the presence of GTP (IC
50
¼ 3 · 10
)3
and
5 · 10
)4
m in the presence and absence of GTP,
respectively; Fig. 2B). In contrast, the affinity for atro-
pine was not affected by GTP (Fig. 2A). These results
indicate that M
2
in the fusion protein interacts with
GOA-1 as well as with Ga
i1
in a GTP-sensitive manner.
Discussion
In this study, we have shown that the human Ga
i ⁄ o
-
coupled receptor, M
2
, can activate GOA-1, which is
the Ga
i ⁄ o
ortholog in the nematode C. elegans. The
ligand-binding properties of M

o
(accession no.
NM138736) as the most similar Ga in mammals. The
amino acid sequence of GOA-1 is 82.2% identical to
that of Ga
o
. Although the similarity of the aA–aE
region and the region upstream of a4 was relatively
low, the N-terminal region (aN–a1), the aF–aG region
and the C-terminal region (a4–a5) were well conserved
between GOA-1 and Ga
o
[31,32]. The five regions of
the Ga-subunit involved in receptor recognition are
the a2 helix, the b6 ⁄ a5 loop, the a5 helix and the
N- and C- extreme termini [31]. In addition, the a4
helix and a4 ⁄ b6 loop region of Ga
i1
are important for
specific recognition of receptors [33]. The distinct sub-
stitution observed in GOA-1 was relatively rare in
those regions, suggesting that M
2
should interact with
GOA-1 in a similar manner to that of Ga
i ⁄ o
.
The EC
50
value of carbamylcholine for myc-M

(N-D)I3del::Ga
i1
. In addition, the myc-tagged M
2
was reported to be indistinguishable from the unmodi-
fied M
2
in [
3
H]QNB binding [34]. These results suggest
Fig. 3. Effect of carbamylcholine and atropine on [
35
S]GTPcS bind-
ing of myc-M
2
(N-D)I3del::GOA-1. All experiments were performed
in triplicate. Each data point represents the mean ± SEM. (A)
Increase in [
35
S]GTPcS binding by carbamylcholine in the presence
of 10
)6
M GDP. M
2
(N-D)I3del alone caused no increase in
[
35
S]GTPcS binding [25]. (B) Decrease in GDP affinity by carbamyl-
choline. The experiment was performed in the presence of 1 m
M

coupled receptors may be compatible between
mammals and nematodes. As mentioned previously,
overexpression of GOA-1 causes various visible phe-
notypes in C. elegans, suggesting that further in vivo
studies should be performed using C. elegans towards
the use of GPCRs derived from evolutionarily distant
organisms for manipulation of G-protein signalling.
In this study, M
2
was selected as a model of mamma-
lian Ga
i ⁄ o
-coupled receptors mainly due to its conveni-
ence for experimental use, i.e. the abundant expression
using a baculovirus system has been established, and
the pharmacological properties have been revealed in
detail. Here, we evaluate M
2
for the manipulation of
GOA-1 signalling in C. elegans. The natural ligand of
M
2
, acetylcholine, is a neuro- and neuromuscular trans-
mitter not only in mammals, but also in nematodes
[36], suggesting that the use of M
2
is restricted by the
influence of intrinsic acetylcholine. In C. elegans, three
muscarinic acetylcholine receptors have been found
(GAR-1, -2 and -3). Although the pharmacological

mutant, myc-M
2
(N-D)I3del, was
amplified by PCR using the M
2
(N-D)I3del::Ga
i1
expression
construct, pPAK-M
2
–Ga
i1
[25], as a template with the
following primers: M
2
-myc-EcoRI-s, 5¢-CAGAATTCatg
gagcagaagctgatctccgagga ggacctg ctg

GTGAACAACTCCAC
CAACTCCTCCAACAACTCCCTGGCTCTTACAAGTC
CTTATAAGACA-3¢; HsM2-as, 5¢-TTACCTTGTAGCG
CCTATGTTCTTATAATG-3¢. (An engineered EcoRI
recognition site is single-underlined. The start codon is
double-underlined. The modified original start codon of M
2
is dot-underlined. The engineered region containing myc-
epitope tag encoded region is indicated in lower case.)
GOA-1 cDNA was amplified by RT-PCR using total RNA
separated from mix stage of C. elegans as a template with
the following primers: M2-goa1-s, 5¢-

thawed and homogenized in Sf9 buffer (20 mm
Hepes ⁄ KOH, pH 8.0, 1 mm EDTA, 2 mm MgCl
2
,2mm
EGTA, 1 lm pepstatin, 10 lm leupeptin, 0.28 lm E64,
0.2 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluor-
ide) on ice. The homogenate was centrifuged at 150 000 g
for 1 h. The pellet (membrane fraction) was resuspended
in phosphate buffer (137 mm NaCl, 2.7 mm KCl, 8.1 mm
Na
2
HPO
4
, 1.47 mm KH
2
PO
4
, pH 7.44). Protein concentra-
tion was assessed using a BCA assay kit (Pierce, Rock-
ford, IL) and adjusted at 1 mgÆmL
)1
by adding phosphate
buffer.
Western blot
Five microliters of the membrane fraction was resuspended
in 1· SDS ⁄ PAGE loading buffer. SDS ⁄ PAGE was per-
formed using a 3–15% (w ⁄ v) polyacrylamide gel (ATTO,
Tokyo, Japan). Following electrophoresis, the gel was
M. Minaba et al. Human GPCR activates nematode G protein
FEBS Journal 273 (2006) 5508–5516 ª 2006 The Authors Journal compilation ª 2006 FEBS 5513

ceptors. After incubation at 30 °C for 30 min, the reac-
tion was terminated by filtration using a UniFilter-96
(Hewlett Packard, Palo Alto, CA), and rinsed three times
with a KPB buffer (20 mm potassium phosphate, pH 7.0,
0.1 mm NaN
3
) to remove the free labeled compounds.
Scintillation reagent (Microscint20; Hewlett Packard) was
added to each well of the air-dried filter. The radioactiv-
ity of the membrane fraction on the filter was measured
with a scintillation counter (TopCount NXT; Hewlett
Packard).
To estimate the relative affinity of carbamylcholine and
atropine, displacement of [
3
H]QNB binding was estimated.
The membrane fraction containing 10 lg of recombinant
protein was mixed with various concentrations of carba-
mylcholine or atropine in 100 lL of phosphate buffer.
The solution was mixed with 1 nm (final) [
3
H]QNB and
incubated at 30 °C for 30 min. The reaction mixture was
filtered, and the radioactivity was measured as described
previously.
GTPcS-binding assay
The binding reaction was performed in 100 lL of binding
assay buffer (20 mm Hepes ⁄ KOH, pH 8.0, 1 mm EDTA,
160 mm NaCl, and 10 mm MgCl
2

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