muscarinic acetylcholine receptor M2 subtypeFunctional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals Masaomi Minaba1, Susumu Ichiyam
Trang 1muscarinic acetylcholine receptor M2 subtype
Functional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals
Masaomi Minaba1, Susumu Ichiyama2, Katsura Kojima3, Mamiko Ozaki4and Yusuke Kato1
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-gle Ga must couple with many GPCRs [4,5]
GPCR–G protein signalling regulates various
phy-siological functions in a wide variety of organisms
including plants and animals [6–9] Therefore, such
physiological functions can be affected by manipula-tion of the GPCR–G protein signal transducmanipula-tion Our interest is in the use of GPCRs derived from evolutio-narily distant organisms for the manipulation of
G protein signalling GPCRs recognize a wide variety
of ligands Although some ligands are conserved in many organisms (e.g acetylcholine, serotonin), others are recognized in only a few organisms (e.g peculiar peptide hormones) GPCRs recognizing such unique ligands are often found in evolutionarily distant organ-isms If such GPCRs can couple with the target Ga,
we can manipulate the GPCR–G protein signalling of transgenic individuals by using specific ligands that do not activate any receptors in wild-type individuals However, the coupling of GPCRs and G proteins
Keywords
biotechnology; Caenorhabditis elegans;
G protein; muscarinic acetylcholine receptor;
nematodes
Correspondence
Y Kato, Immune Defense Unit, National
Institute of Agrobiological Sciences,
Tsukuba, Ibaraki 305-8634, Japan
Fax ⁄ Tel: +81 29 838 6059
E-mail: kato@affrc.go.jp
(Received 18 August 2006, revised 12
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, Gai⁄ o ortho-log in the nematode Caenorhabditis elegans, with the human muscarinic acetylcholine receptor M2 subtype (M2), which is one of the mammalian
Gai⁄ o-coupled receptors GOA-1 and M2were 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 ([35S]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 M2–Gai⁄ o coup-ling These results indicate that the M2activates GOA-1 as well as Gai ⁄ o
Abbreviations
EC50, 50% effective concentration; Ga, Gb and Gc, heterotrimeric G protein alpha, beta and gamma subunits; GPCR, G protein-coupled receptor; GTPcS, guanosine 5¢-[3-O-thio]triphosphate; IC 50 , 50% inhibitory concentration; M 2 , muscarinic acetylcholine receptor M 2 subtype; NMS, N-methylscopolamine; QNB, L -quinuclidinyl benzilate.
Trang 2derived from evolutionarily distant organisms has not
been systematically examined
Mammalian Ga of heterotrimeric G protein is
categ-orized into four groups (Gas, Gai ⁄ o, Gaq ⁄ 11and Ga12)
[10] Gaois a member of the Gai ⁄ oclass Caenorhabditis
elegans, a nematode, is a genetic model organism widely
used in laboratories [11] The whole genome sequence of
C eleganshas been determined in multicellular
organ-isms [12] In the genome of C elegans, 21 Ga have been
found [13,14] Although some Ga appear to be unique
in nematodes, orthologs of mammalian Gas, Gaq, Ga12
and Gai⁄ ohave also been identified GOA-1 is the Gai⁄ 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
adaptation [19] The function of GOA-1 in neurons is
partly explained as antagonizing EGL-30 (Gaq
ortho-log) [20,21] Furthermore, GOA-1 controls embryonic
spindle positioning in single-cell embryos [22] In the
embryo, GOA-1 is activated by cytoplasmic guanine
exchange factor-like protein, RIC-8, independently of
GPCR [22] However, there is no experimental evidence
for GOA-1 activation by GPCRs
In this study, we tested the functional coupling of the
mammalian Gai⁄ o-coupled receptor, human M2, to the
nematode GOA-1 as a model of manipulation using
GPCRs derived from evolutionarily distant organisms
The M2receptor has been best characterized as a Gai⁄ o
-coupled receptor since its primary structure
determin-ation in 1986 [23] Although Gb and Gc are essential for
Ga activation by GPCR [10], GPCR can activate Ga
without Gb and Gc in some GPCR::Ga fusion proteins
[24] Muscarinic-agonist-dependent Ga activation can
be detected in an M2::Gai1fusion protein [25] A
large-scale preparation of the fusion protein has been
estab-lished using a baculovirus expression system [25]
Therefore, M2 receptor is one of the best models for
mammalian Gai⁄ 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 M2 with GOA-1 We prepared an
M2mutant::GOA-1 fusion protein and directly assessed
the muscarinic-ligand-dependent activation of GOA-1
Results
Expression of M2::GOA-1 fusion protein
The human M2 receptor is a Gai⁄ o-coupled receptor
To test whether M2 can activate GOA-1, a fusion
protein of M2 mutant and GOA-1, myc-M2 (N-D)I3-del::GOA-1, was expressed in the insect culture cell Sf21 using a baculovirus expression system (Fig 1A)
A
B
Fig 1 Expression of myc-M2(N-D)I3del::GOA-1 (A) Diagram of expression construct GOA-1 was directly fused at the C-terminus
of M2 A myc-epitope tag was added at the N-terminus of M2 To prevent rapid degradation, the central part of the third intracellular loop of M 2 was deleted Asn at putative N-glucosylation sites near the N-terminus of M 2 were mutated to Asp to avoid various migra-tions in western blot analyses (B) Western blot analysis of myc-M 2 (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.
Trang 3In this recombinant protein, GOA-1 was directly fused
at the C-terminus of M2 A myc-epitope tag was added
at the N-terminus of M2 To prevent rapid
degrad-ation, the central part of the third intracellular loop of
M2 was deleted [26] Asn at putative N-glycosylation
sites near the N-terminus of M2 was mutated to Asp
to avoid diversified migration in western blot analyses
[27] These modifications to M2 do not interfere with
the M2–Gai⁄ o interaction [28] Membrane fractions of
transfected cells were subjected to western blot analysis
with anti-myc serum (Fig 1B) A major band (78 kDa)
was detected specifically in transfected cells expressing
the recombinant protein This is almost identical to the
calculated molecular mass (77 660.17 Da), suggesting
that myc-M2(N-D)I3del::GOA-1 was expressed over
the detection sensitivity in the western blot analysis
and well transferred in the membrane fraction
Binding properties of muscarinic ligands with
M2::GOA-1 fusion protein
The binding properties of muscarinic ligands were
examined to reveal whether the M2 receptor in
myc-M2(N-D)I3del::GOA-1 was functional The membrane
fraction expressing myc-M2(N-D)I3del::GOA-1 showed
high-affinity binding to the radiolabeled ligands
l-quinuclidinyl benzilate ([3H]QNB) and
N-methyl-scopolamine ([3H]NMS) (Fig 2A) This binding was
abolished by addition of the full antagonist, atropine
In addition, the 50% inhibitory concentration (IC50)
for [3H]QNB displacement was estimated for atropine (5· 10)8m), and the full agonist, carbamylcholine (5· 10)4m) in the absence of GTP (Fig 2B) These
IC50 values are very like those with M2(N-D)I3del::
Gai1 (5.0· 10)8m for atropine, and 3.3· 10)4m for carbamylcholine) [25] These results indicate that M2
in the GOA-1 fusion protein is functional, and the ligand-binding properties agree with that of the Gai1 fusion protein
Activation of GOA-1 by muscarinic agonists Agonist-bound GPCRs are considered to interact with
G proteins This interaction causes a decrease in the affinity for GDP of Ga and the subsequent substitu-tion of GDP by GTP [10] Such an agonist-dependent decrease in the affinity for GDP can be detected as the increase in binding of the nonhydrolysable GTP ana-log guanosine 5¢-[3-O-thio]triphosphate (GTPcS) This agonist-dependent decrease of GDP affinity has also been demonstrated in membrane preparations expres-sing the M2::Gai1 fusion protein [25] Here, similar binding properties of GDP and GTPcS were observed
in the membrane preparations expressing myc-M2 (N-D)I3del::GOA-1 The binding of GTPcS was increased by stimulation of myc-M2(N-D)I3del:: GOA-1 with carbamylcholine in a dose-dependent manner (Fig 3A) The 50% effective concentration (EC50) value was estimated to be 10)5m 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-M2(N-D)I3del::GOA-1 The membrane fraction containing 10 lg 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)13M or carbamylcholine ¼ 10)7M , respectively.
Trang 4atropine (Fig 3A), suggesting that carbamylcholine
induced the substitution for GTPcS in GOA-1 The
IC50value of the displacement of GTPcS by GDP was
3· 10)6m with 0.2 mm atropine, and 10)4m without
atropine in the presence of 1 mm carbamylcholine,
which induced the maximum increase in GTPcS
bind-ing (Fig 3B), suggestbind-ing that carbamylcholine caused
the decrease of GDP affinity in GOA-1 These results
indicate that M2 functionally couples with GOA-1 as
well as Gai1
GTP affects affinity of muscarinic ligands
The affinity of M2 for agonists, but not for
antago-nists, decreases on interaction with Gai⁄ o in the
pres-ence of guanine nucleotides [28–30] The affinity for
carbamylcholine of myc-M2(N-D)I3del::GOA-1 dec-reased in the presence of GTP (IC50¼ 3 · 10)3 and
5· 10)4m 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 M2 in the fusion protein interacts with GOA-1 as well as with Gai1in a GTP-sensitive manner
Discussion
In this study, we have shown that the human Gai⁄ o -coupled receptor, M2, can activate GOA-1, which is the Gai⁄ o ortholog in the nematode C elegans The ligand-binding properties of M2 mutant::GOA-1 fusion protein were similar to those of Gai⁄ o fusion protein
In addition, GTP causes the decrease of affinity for carbamylcholine to M2 These properties are almost identical to those of M2–Gai ⁄ o coupling, suggesting that the function of GOA-1 and Gai ⁄ o was evolutio-narily conserved in the coupling with M2
These results also indicate that the distinct amino acids in GOA-1 are neutral for coupling to M2 An alignment of GOA-1 and other M2-coupled Ga pro-teins is shown in Fig 4 GOA-1 is the ortholog of mammalian Gai⁄ o in C elegans BLAST database searches identified the human Gao (accession no NM138736) as the most similar Ga in mammals The amino acid sequence of GOA-1 is 82.2% identical to that of Gao 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 Gao [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 Gai1are important for specific recognition of receptors [33] The distinct sub-stitution observed in GOA-1 was relatively rare in those regions, suggesting that M2 should interact with GOA-1 in a similar manner to that of Gai⁄ o
The EC50 value of carbamylcholine for myc-M2 (N-D)I3del::GOA-1 in GTPcS binding was estimated to
be 10)5m in the presence of 1 lm GDP and 10 mm MgCl2 This value is greater than that of the fusion protein to Gai1 without the N-terminal myc-tag,
M2(N-D)I3del::Gai1 (2.6· 10)7m), under the same experimental conditions [25] The IC50 values of atro-pine and carbamylcholine for myc-M2(N-D)I3del:: GOA-1 in [3H]QNB displacement was like those of
M2(N-D)I3del::Gai1 In addition, the myc-tagged M2
was reported to be indistinguishable from the unmodi-fied M2 in [3H]QNB binding [34] These results suggest
Fig 3 Effect of carbamylcholine and atropine on [35S]GTPcS
bind-ing of myc-M2(N-D)I3del::GOA-1 All experiments were performed
in triplicate Each data point represents the mean ± SEM (A)
Increase in [35S]GTPcS binding by carbamylcholine in the presence
of 10)6M GDP M2(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
carbamylcholine Dotted line, in the presence of 0.2 m M atropine;
solid line, in the absence of atropine.
Trang 5that the addition of myc-tag should not affect the
lig-and-binding properties of M2 The mechanism of this
difference in EC50values remains to be elucidated
To date, GOA-1 activation has been reported only
in an RIC-8-dependent and GPCR-independent man-ner using the GTPcS-binding experiment Here, we
Fig 4 Alignment of Ga activated by M2 Secondary structures are indicated [32] The distinct amino acid residues only observed in GOA-1 are represented as inversed characters (gray, distinct but similarity was conserved among all Ga; black, distinct and not similar).
Trang 6represented the GPCR-dependent GOA-1 activation.
Therefore, both GPCR-dependent and -independent
activation of GOA-1 have been experimentally
evi-denced
C elegans is the best genetic model The generation
of transgenic C elegans has been established and is
easier than that of other animal models [35] The
results of this study indicate that the Gai⁄ o⁄
GOA-1-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, M2was selected as a model of
mamma-lian Gai⁄ 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 M2 for the manipulation of
GOA-1 signalling in C elegans The natural ligand of
M2, acetylcholine, is a neuro- and neuromuscular
trans-mitter not only in mammals, but also in nematodes
[36], suggesting that the use of M2 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
properties of GAR-1 and -2 are clearly distinct from
those of mammals [37,38], that of GAR-3 is
compar-able (e.g the muscarinic agonist, oxotremorine, is
effective on GAR-3, but not on GAR-1 and -2) [39],
suggesting that the manipulation of GOA-1 signalling
by M2using muscarinic agents may be accompanied by
affecting GAR-3 in C elegans However, gar-3 is
expressed only in the pharynx and controls pharyngeal
pumping [40], indicating that the side effect of the
acti-vation of GAR-3 is limited Furthermore, the
pheno-type of gar-3 loss-of-function mutants is almost
wild-type with the exception of a faster pharyngeal pumping
rate [40], suggesting that the side effect of GAR-3
acti-vation may be avoided using M2transgenic worms in a
gar-3mutant background In conclusion, M2is a good
candidate for the manipulation of GOA-1 signalling in
C elegansunder carefully controlled conditions
Experimental procedures
Expression of myc-M2(N-D)l3del::GOA-1 fusion
protein
The cDNA-encoding M2 mutant, myc-M2(N-D)I3del, was
amplified by PCR using the M2(N-D)I3del::Gai1expression
construct, pPAK-M2–Gai1 [25], as a template with the following primers: M2-myc-EcoRI-s, 5¢-CAGAATTCatg gagcagaagctgatctccgaggaggacctgctg
-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 M2
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¢-CATTATAAGA ACATAGGCGCTACAAGGATGGGTTGTACCATGTC ACAGGAAG-3¢; M2-goa1-PstI-as, 5¢-CCAATGCATTGG TTCTGCAGTTAATACAAGCCGCATCCACGAAGA-3¢ (An engineered PstI recognition site is single-underlined The overlapping region to the C-terminus of M2is double-underlined.) The cDNAs encoding M2(N-D)I3del and GOA-1 were fused by a fusion PCR using the overlapping region The fusion cDNA contains a myc-epitope tag (EQ-KLISEEDL) and an EcoRI recognition site at the 5¢-end and a PstI recognition site at 3¢-end The fusion PCR prod-uct was cloned into a baculovirus transfer vector, pFAST-Bac1 (Invitrogen, Carlsbad, CA) using the engineered restriction sites Recombinant baculoviruses were generated
in Sf21 insect culture cells by using a Bac-to-Bac Baculo-virus Expression Kit (Invitrogen) The conditioned medium containing the recombinant viruses was directly used for the production of recombinant protein Sf21 cells were grown at 28C to an 80% confluent monolayer and infec-ted with recombinant viruses The cells were harvesinfec-ted at
48 h after infection and stored at)80 C
Membrane preparation Frozen myc-M2(N-D)I3del::GOA-1 expressed cells were thawed and homogenized in Sf9 buffer (20 mm Hepes⁄ KOH, pH 8.0, 1 mm EDTA, 2 mm MgCl2, 2 mm 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
Na2HPO4, 1.47 mm KH2PO4, 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
Trang 7washed in Towbin transfer buffer (25 mm Tris⁄ HCl,
192 mm glycine, 20% methanol, pH 8.3) After soaking the
gel in Towbin transfer buffer containing 0.05% SDS for
15 min to facilitate the transfer efficiency of large
mole-cules, proteins were transferred onto a polyvinylidene
fluo-ride membrane (Immobilon P; Millipore, Billerica, MA)
using a semidry blotting equipment (Trans Blot SD;
Bio-Rad, Hercules, CA) After transfer, the membrane was
incubated in TBS-T buffer (20 mm Tris⁄ HCl, 140 mm
NaCl, 1% Tween 20, pH 7.5) containing 5% skimmed milk
as a blocking agent for 1 h The blocked membrane was
incubated with an alkaline phosphatase conjugated
mono-clonal antibody against myc (Invitrogen) for 1 h Using an
AP conjugate substrate kit (Bio-Rad), myc-tagged
recom-binant proteins were detected as deep violet bands
Ligand-binding assay
Binding of the radiolabeled muscarinic partial antagonists,
[3H]QNB and [3H]NMS, was assessed The membrane
fraction containing 10 lg recombinant protein was added
to 100 lL of the phosphate buffer containing 0.1 nm
[3H]QNB or 4 nm [3H]NMS in the presence or absence
of 0.2 mm atropine, a full antagonist for muscarinic
re-ceptors After incubation at 30C for 30 min, the
reac-tion was terminated by filtrareac-tion 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 NaN3) 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 [3H]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) [3H]QNB and
incubated at 30C 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 MgCl2) containing 0.1 nm
[35S]GTPcS Various concentrations of GDP, agonists and
antagonists were added depending on the experimental aim
After the addition of 10 lg of membrane fraction, the
reac-tion mixture was incubated at 30C for 1 h The reaction
mixture was filtered, and the radioactivity was measured as
described previously
Computer-assisted sequence analysis BLAST database searches were performed via http:// www.ncbi.nlm.nih.gov/BLAST/ The molecular mass was estimated via http://usexpasy.org/tools/pi_tool.html
Acknowledgements
We are grateful to Professor Tatsuya Haga (Gakushuin University) for helpful suggestions and critical reading of this manuscript This work was supported by the program for Promotion of Basic Research Activities for Innovative Biosciences, Japan
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