Biochem. J. (2010) 432, 267–273 (Printed in Great Britain)
267
doi:10.1042/BJ20100617
Guanylate cyclase-G, expressed in the Grueneberg ganglion olfactory
subsystem, is activated by bicarbonate
Ying-Chi CHAO*†1 , Chien-Jui CHENG‡1 , Hsiu-Ting HSIEH†, Chih-Ching LIN†, Chien-Chang CHEN† and Ruey-Bing YANG*†§2
*Molecular Medicine Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan, †Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan,
‡Graduate Institute of Clinical Medicine and Department of Pathology, College of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan, and §Institute of Pharmacology,
School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
GC (guanylate cyclase)-G is the most recently identified member
of the receptor GC family. However, the regulation of its
activity and protein expression in the mammalian olfactory
system remains unclear. In the present study, we used a GC-Gspecific antibody to validate that the GC-G protein is expressed
in Grueneberg ganglion neurons, a newly recognized olfactory
subsystem co-expressing other cGMP signalling components such
as the cGMP-regulated PDE2A (phosphodiesterase 2A) and the
cGMP-gated ion channel CNGA3 (cyclic nucleotide-gated cation
channel α-3). Further molecular and biochemical analyses showed
that heterologously expressed GC-G protein, specifically the Cterminal cyclase domain, was directly stimulated by bicarbonate
in both in vivo cellular cGMP accumulation assays in human
embryonic kidney-293T cells and in vitro GC assays with a
purified recombinant protein containing the GC domain. In
addition, overexpression of GC-G in NG108 neuronal cells
resulted in a CO2 -dependent increase in cellular cGMP level that
could be blocked by treatment with acetazolamide, an inhibitor of
carbonic anhydrases, which implies that the stimulatory effect
of CO2 requires its conversion to bicarbonate. Together, our
data demonstrate a novel CO2 /bicarbonate-dependent activation
mechanism for GC-G and suggest that GC-G may be involved in
a wide variety of CO2 /bicarbonate-regulated biological processes
such as the chemosensory function in Grueneberg ganglion
neurons.
INTRODUCTION
CO2 detection, responses that require the activity of type II
CA (carbonic anhydrase) to catalyse the conversion of CO2 to
bicarbonate [9,10]. Advanced biochemical studies demonstrated
that bicarbonate can directly activate the cGMP-generating
activity of GC-D [10,11].
GC-G is the latest identified member of the GC family [12–14].
Use of a commercially available antibody revealed anti-GCG immunoreactive staining localized in the GG neurons of
the olfactory subsystem [15]. However, the specificity of this
antibody (i.e. its cross-reactivity with other receptor GCs) has
not been comprehensively examined. Therefore, whether GCG is indeed expressed in olfactory tissues and how GC-G is
regulated in GG neurons remain unclear. In the present study, we
produced and used an anti-GC-G-specific antibody to validate that
GC-G protein is expressed in GG neurons and demonstrated that
bicarbonate activates GC-G by directly acting on the cytosolic
cyclase catalytic domain of GC-G.
GCs (guanylate cyclases), which produce the intracellular second
messenger cGMP, mediate a broad spectrum of physiological
processes, such as arterial blood pressure and volume
homoeostasis, endochondral ossification, intestinal electrolyte
and fluid transport, epithelial cell growth and differentiation,
and phototransduction in the retina [1]. However, knowledge of
the complete range of cellular functions modulated by cGMP is
expanding and perhaps not fully understood. In mammals, seven
isoforms of GCs were designated GC-A to GC-G in order of
their discovery [1]. These GCs share an organized protein domain
structure of at least four recognizable motifs: an extracellular
ligand-binding domain, followed by a single transmembrane
segment and a cytoplasmic region that can be further subdivided
into a protein kinase-like domain and a C-terminal cyclase
catalytic domain. GCs can be activated by extracellular peptide
ligands [1], weakly by intracellular calcium-binding proteins [2,3]
or via the small GTPase Rac and p21-activated kinase pathway
[4].
The murine olfactory system is composed of at least four
anatomically separate subsystems: the main olfactory epithelium,
vomeronasal organ, septal organ and GG (Grueneberg ganglia);
each subsystem is likely to have distinct functions [5,6]. GC-D
was shown to be expressed in a small subset of rat main olfactory
epithelial neurons. Previous genetic and electrophysiological
studies demonstrated that GC-D-positive OSNs (olfactory sensory
neurons) are stimulated by the peptides uroguanylin and guanylin
[7,8]. In addition, GC-D-positive OSNs are responsible for
Key words: bicarbonate, cGMP, guanylate cyclase (GC),
Grueneburg ganglion (GG), receptor, signal transduction.
EXPERIMENTAL
Reagents
All chemicals were of reagent grade from Sigma. Anti-FLAG
M2 and anti-Myc 9E10 monoclonal antibodies were purchased
from Sigma and Covance respectively. Anti-CA II antiserum
was from Santa Cruz Biotechnology. The vector-based shRNA
(short hairpin RNA) to knock down mouse GC-G (clone
TRCN0000012167) or a non-targeting shRNA for GFP (green
Abbreviations used: AZ, acetazolamide; CA, carbonic anhydrase; CNGA3, cyclic nucleotide-gated cation channel α-3; FL, full-length; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GC, guanylate cyclase; GFP, green fluorescent protein; GG, Grueneberg ganglion; GST, glutathione
transferase; HEK-293T, human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40); OSN, olfactory sensory neuron;
PDE, phosphodiesterase; RNAi, RNA interference; RT–PCR, reverse transcription–PCR; sAC, soluble adenylate cyclase; shRNA, short hairpin RNA.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
268
Y.-C. Chao and others
fluorescent protein; clone TRCN0000072178) were obtained
from The RNAi Consortium [16].
Cell culture and transfection
HEK-293T [human embryonic kidney-293 cells expressing the
large T-antigen of SV40 (simian virus 40)] and NG108 neuronal
cells were maintained in DMEM (Dulbecco’s minimal essential
medium) supplemented with 10 % heat-inactivated fetal bovine
serum, 100 units/ml penicillin and 100 μg/ml streptomycin at
37 ◦ C in an atmosphere of 5 % CO2 . Cells were transfected using
LipofectamineTM 2000 (Invitrogen).
Construction of expression plasmids
The FLAG-tagged FL (full-length) GC-G expression plasmid
(FLAG–GC-G) was constructed as described previously [13]. The
ECD+KLD or CYC expression constructs code for a mutant
protein lacking residues 44–455 and 556–833 or residues 834–
1003 respectively. The CYC expression plasmid encodes only
the C-terminal cyclase domain (residues 692–1100). All other
expression plasmids (e.g. FLAG–GC-A to E) were constructed in
a similar fashion in the pFLAG-CMV1 vector (Sigma).
GC activity assay
The concentration of cGMP was measured by the CatchPoint
cGMP fluorescent assay kit (Molecular Devices). To demonstrate
the biocarbonate activation, transfected cells were cultured in
bicarbonate-free L-15 medium (GIBCO) in a CO2 -deprived
incubator for 24 h. Cells were then collected into new tubes and
stimulated with bicarbonate at the indicated concentrations for
20 min at 37 ◦ C together with 1 mM isobutylmethylxanthine [a
PDE (phosphodiesterase) inhibitor]. Cell lysates were prepared,
and cellular cGMP content was determined according to the
manufacturer’s instructions (Molecular Devices).
For the in vitro GC activity assay, purified recombinant GST–
GC-G-CYC protein (1 μg) was mixed with 50 μl of 2×
reaction buffer (50 mM Hepes, pH 7.4, 300 mM NaCl, 2 mM
isobutylmethylxanthine, 4 mM ATP, 4 mM GTP, 1 mg/ml BSA,
10 mM MgCl2 and 20 mM sodium azide) and the indicated
concentrations of bicarbonate to a final volume of 100 μl. After
incubation for 20 min at 37 ◦ C, the reaction was stopped by
adding 250 μl of ice-cold 100 % ethanol. After centrifugation
(3000 g, 5 min, 4 ◦ C), the supernatant was dried in a speed-vacuum
concentrator, and cGMP content was measured using the cGMP
assay kit.
Cell imaging
Establishment of stable cell lines expressing GC-G
HEK-293T cells were co-transfected with the plasmid encoding
FLAG–GC-G and the pcDNA4 vector (Invitrogen) containing
the antibiotic zeocin resistance gene. Two days post-transfection,
stable GC-G-expressing cells were selected with 400 μg/ml
zeocin (Invitrogen). Zeocin-resistant cell clones were examined
by anti-FLAG Western blotting to determine the expression of the
FLAG-tagged GC-G, and the positive clones were amplified for
further analysis.
Immunoprecipitation and Western blot analysis
Two days after transfection, cell lysates were clarified by
centrifugation at 10 000 g for 20 min at 4 ◦ C. Samples underwent
immunoprecipitation, then Western blot analysis was performed
as previously described [17].
Immunohistochemistry
All animal procedures were performed according to the
protocols approved by the Institutional Animal Care and
Utilization Committee, Academia Sinica. Heads of 1-day-old
mice (C57BL/6) were dissected, fixed in 4 % paraformaldehyde
overnight at 4 ◦ C, and embedded in paraffin. Sections were cut at
5 μm. Anti-GC-G immunohistochemical analysis was performed
as described previously [18].
Preparation of GST (glutathione transferase)-fusion proteins
GST-fusion proteins were constructed in the expression vector
pGEX-4T (GE Healthcare). The PCR fragment encoding the
GC-G cyclase domain (amino acids 849–1048) was ligated into
the vector at the BamRI/XhoI sites to generate the GST–GCG-CYC fusion protein. The fusion protein was expressed in
Escherichia coli and purified from the soluble fraction of the
bacterial lysate with glutathione–Sepharose beads according to
the manufacturer’s instructions (GE Healthcare).
c The Authors Journal compilation c 2010 Biochemical Society
HEK-293T cells were plated on a 35-mm glass-bottom dish. One
day later, cells were transiently transfected with use of FuGENETM
6 (Roche) and an expression plasmid encoding a fluorescent
indicator cGMP (α-FlincG) [19] alone or together with another
construct encoding GC-G. After 24 h, transfected cells were
stimulated with 30 mM NaHCO3 in culture medium for 15 min.
Imaging experiments involved spinning disk confocal microscopy
(Ultra-View, PerkinElmer). Cells were imaged on an inverted
microscope (IX71, Olympus) with a 60× oil-immersion objective
and maintained at 37 ◦ C during the experiments. Intracellular
cGMP level reflected by the intensity of the fluorescent cGMP
biosensor was recorded by ratiometric excitation at 410 and
480 nm and emission at 510 nm [19]. Images and data were
analysed using of MetaMorph Software (Molecular Devices).
Statistical analysis
Data are expressed as means +
− S.E.M. Differences between
groups were analysed by unpaired Student’s t test. P < 0.05 was
considered statistically significant.
RESULTS
Specificity of the anti-GC-G antibody and expression of GC-G
in GG neurons
To validate the specificity of the anti-GC-G antibody we
developed previously [12], we first heterologously expressed
each isoform of receptor GCs (GC-A to G) in HEK293T cells. Western blot analysis revealed that the anti-GCG antibody specifically recognized the recombinant GC-G
protein, but not other receptor GCs (Figure 1A). We then
examined the expression of GC-G by immunohistochemistry
in the frontal nasal sections containing the GG neurons of
neonatal mice using this anti-GC-G-specific antibody. The
GG neurons were positive for anti-GC-G immunoreactivity
(Figures 1B and 1C). Pre-incubation of anti-GC-G antibody
with its corresponding immunogen or omission of the primary
antibody resulted in no staining (results not shown), thus
A novel bicarbonate-sensitive receptor guanylate cyclase
Figure 2
Figure 1 Specificity of the anti-GC-G antibody and expression of GC-G in
GG neurons
(A) Specificity of the anti-GC-G antibody. The anti-GC-G polyclonal antibody was raised in
rabbits against the extracellullar domain of GC-G as described previously [12]. The anti-GC-G
antibody could specifically detect the recombinant FLAG-tagged GC-G protein, but not other
receptor GC isoforms expressed in HEK-293T cells. As a loading control, anti-FLAG antibody
was used to confirm the protein expression of FLAG-tagged GC receptors. Molecular mass
is indicated in kDa to the left. WB, Western blot analysis. (B and C) GC-G expression in GG
neurons. Cross-sections of the mouse nasal vestibule containing the GG neurons were stained
with the anti-GC-G-specific antibody. GC-G immunoreactivity was predominantly detected in
GG neurons (arrows). Scale bar: 200 μm in (B); 50 μm in (C).
demonstrating the specificity of anti-GC-G immunostaining in
the GG neurons.
Bicarbonate increases GC-G activity in cells
Because bicarbonate can directly stimulate the activity of GC-D
in a subset of OSNs [9,10], we investigated whether the cyclase
activity of GC-G expressed in GG is regulated by bicarbonate in
cells. After transient transfection, the expression of the FLAG–
GC-G was confirmed by Western blot analysis (Figure 1A) and
shown to be targeted on the cell surface of HEK-293T cells by flow
269
GC-G expressed in HEK-293T cells is activated by bicarbonate
(A) Cell-surface expression of GC-G. HEK-293T cells were transfected with an empty vector or an
expression plasmid encoding the FL GC-G protein. A FLAG epitope tag was added immediately
after the signal peptide sequence at the N-terminus for easy detection (FLAG.GC-G). Two
days after transfection, cells were detached and stained with anti-FLAG antibody to determine
the cell-surface expression by FACS analysis. (B) Bicarbonate stimulated the cyclase activity
of GC-G expressed in HEK-293T cells. At 24 h after transfection, the vector-transfected or
GC-G-expressing cells were stimulated with the indicated concentrations of NaHCO3 for 20 min.
Cellular cGMP content was measured as described in the Experimental section. (C) Effect of
NaHCO3 , KHCO3 and NaCl on GC-G activation. After stimulation of GC-G-expressing HEK-293T
cells with the indicated ions (30 mM) for 20 min, intracellular cGMP content was measured.
Results are expressed as means +
− S.E.M. from three experiments.
cytometry (Figure 2A). Because bicarbonate in the extracellular
medium can be transported into cells within minutes and has been
used to study its effect on the cyclase activity of sAC (soluble
adenylate cyclase) expressed in HEK-293T cells [20], we adopted
a similar system to test the bicarbonate effects on GC-G activity.
The addition of NaHCO3 to the extracellular medium stimulated
cellular cGMP accumulation in HEK-293T cells expressing
GC-G, but not in vector-transfected cells (Figure 2B). This
activation was dose-dependent for NaHCO3 , with a significant
stimulation at as low as 5 mM and saturation at approx. 30 –
50 mM. This observation agrees with previous studies of the
effective bicarbonate concentration on sAC or GC-D [10,11,20]
and is within the physiological ranges of intracellular bicarbonate
levels for various cellular systems [20,21]. In addition, GC-G
c The Authors Journal compilation c 2010 Biochemical Society
270
Y.-C. Chao and others
Figure 4
activity
Fluorescence imaging showing that bicarbonate stimulates GC-G
HEK-293T cells were transfected with an expression plasmid encoding a fluorescent indicator
of cGMP (α-FlincG) [19] alone (Control) or with an expression construct expressing GC-G.
Fluorescence signal was examined after stimulation with 30 mM bicarbonate (NaHCO3 ; arrow)
in HEK-293T cells co-expressing GC-G and α-FlincG and in cells expressing α-FlincG alone.
Figure 3 RNAi-mediated knockdown of GC-G reduced bicarbonatestimulated cGMP production in a GC-G stable cell line
The parental HEK-293T cells or a GC-G stable cell line was transfected with the indicated
GFP (Control) or GC-G-specific RNAi knockdown plasmid respectively. The knockdown
efficiency or specificity of GC-G was confirmed by anti-FLAG–GC-G or anti-GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) Western blotting analysis (bottom panel). Two
days after transfection, bicarbonate stimulation and cellular cGMP content was performed and
measured as described in the Experimental section.
activity was stimulated equally well by NaHCO3 and KHCO3 ,
but not NaCl (Figure 2C), which suggests that GC-G activation
was specific to the bicarbonate ion and excluded both Na+ ions
and altered ionic strength as regulators of GC-G activity.
Consistent with this finding, a GC-G-specific sequence of
RNAi (RNA interference)-mediated silencing effectively reduced
the GC-G protein expression (> 80 % suppression of protein
expression), which concomitantly diminished not only the basal
but also bicarbonate-stimulated cGMP production in the GCG stable cell line. Together, these results suggest that GC-G is
indeed the main factor regulated by bicarbonate in a cell system
(Figure 3). Furthermore, bicarbonate-stimulated intracellular
cGMP production by GC-G could be dynamically monitored by a
fluorescent indicator of cGMP (α-FlincG) (Figure 4) [19]. These
results suggest that GC-G cyclase activity can be activated by
bicarbonate in a cellular context in the absence of any factor
specific to GG neurons.
The GC domain is essential for bicarbonate stimulation
To investigate which domain is functionally involved in
bicarbonate activation, we engineered three additional deletion
constructs expressing (i) the mutant GC-G protein without the
extracellular and kinase-like domains (ECD+KLD); (ii) the
mutant protein lacking the functional cyclase domain (CYC);
or (iii) the protein fragment containing only the C-terminal cyclase
domain (CYC) (Figure 5A). Western blot analysis confirmed that
all of these mutant proteins were expressed in HEK-293T cells
(Figure 5B). Furthermore, the addition of bicarbonate-stimulated
cellular cGMP production in HEK-293T cells expressed the
ECD+KLD protein to the same degree as the FL GC-G protein
(Figure 5C), which suggests that the extracellular and kinase-like
domains of GC-G are not important for bicarbonate activation.
However, deletion of the functional cyclase domain in the CYC
c The Authors Journal compilation c 2010 Biochemical Society
protein completely abolished the bicarbonate-stimulated cGMP
increase. Likewise, the CYC protein containing only the Cterminal cyclase catalytic domain was responsive to bicarbonate
activation. Together, these data demonstrate that the cyclase
domain of GC-G is critical for bicarbonate sensing and activation,
at least when overexpressed in HEK-293T cells.
Recombinant protein containing the GC-G GC domain can be
directly stimulated by bicarbonate
To validate whether bicarbonate directly acts on the cyclase
domain of GC-G and to eliminate further the possibility of yetunknown cellular factors involved in activation, we produced and
purified the recombinant GST-fusion protein containing the GC-G
cyclase domain (residues 849–1078) (Figure 6A) to examine the
effects of bicarbonate. The addition of bicarbonate significantly
enhanced the cyclase activity of the purified protein in a dosedependent manner (Figure 6B), which is similar to the bicarbonate
effects on GC-G expressed in the cells. The median effective
concentration (EC50 ) of bicarbonate activation was ∼ 15 mM,
which is within the range of the physiological concentrations of
bicarbonate [20]. Most importantly, the bicarbonate stimulation
was not caused by alteration in pH value of the reaction solution
after the addition of bicarbonate, because neither the recombinant
GC-G-CYC protein alone nor bicarbonate-stimulated cyclase
activity of purified GC-G-CYC protein was sensitive to pH
changes ranging from 7.2 to 7.8 (Figure 6C). Together, our results
suggest that bicarbonate can directly activate the cyclase domain
of GC-G without any additional factor from cells.
CO2 stimulates the cyclase activity of GC-G in neuronal cells
Because bicarbonate can be catalytically converted from CO2
by CAs under physiological conditions, we next tested whether
GC-G could be regulated by CO2 in a cellular context. We chose
NG108 neuronal cells for this study, because these cells contain
low basal cGMP level and express both mRNA and protein
levels of type II CA (Figures 7A and 7B). Transfection of the
GC-G expression plasmid resulted in a 3-fold increase of cellular
cGMP level, as compared with vector-transfected neuronal cells
cultured without CO2 (Figure 7C). However, exposure to CO2
further enhanced the cGMP levels in these GC-G-expressing
A novel bicarbonate-sensitive receptor guanylate cyclase
271
Figure 5 The GC catalytic domain of GC-G is required for bicarbonate
activation
(A) Domain structure of FL GC-G and its deletion constructs. A FLAG epitope tag was added at
the N-terminus for easy detection. The ECD+KLD or CYC mutant protein lacks amino acids
44–455 and 556–833 or amino acids 834–1003 respectively. The CYC mutant protein contains
the C-terminal cyclase region (amino acids 692–1100). CYC, cyclase catalytic domain; ECD,
extracellular domain; KLD, kinase-like domain; SP, signal peptide; TM, transmembrane region.
(B) Expression of GC-G FL and deletion constructs. Two days after transfection, cell lysates
were immunoprecipitated, then examined by Western blot analysis with anti-FLAG antibody
used to confirm the expression of the indicated FL GC-G or mutant proteins. (C) A functional
cyclase domain of GC-G is critical for bicarbonate stimulation. HEK-293T cells expressing the
indicated GC-G constructs were treated or not with bicarbonate (NaHCO3 ; 30 mM) for 20 min.
Intracellular cGMP content was measured as described in the Experimental section.
cells (Figure 7C). Interestingly, this CO2 -induced cGMP increase
was completely blocked by treatment with AZ (acetazolamide),
an inhibitor of CAs.
DISCUSSION
In the present study, we developed a GC-G-specific antibody
to validate the unique expression of GC-G in GG neurons and
revealed bicarbonate as a novel activator of GC-G activity in
HEK-293T cells. However, the structure and molecular basis for
the interaction between bicarbonate and the GC-G cyclase domain
remains to be further elucidated. Interestingly, CO2 increased the
Figure 6
GC-G
Bicarbonate directly activates the recombinant cyclase domain of
(A) Construction and purification of the GST-fusion protein containing the GC-G cyclase domain.
Upper panel: diagram of GST fusion protein GST–GC-G-CYC (amino acids 849–1078). Lower
panel: the GST–GC-G-CYC fusion protein purified from the soluble fraction of bacterial lysates
with glutathione–Sepharose beads was analysed by SDS/PAGE and stained with Coommassie
Brilliant Blue. Molecular masses are indicated to the left in kDa. (B) Dose-dependent activation
of purified GC-G cyclase domain by bicarbonate. Purified recombinant GST–GC-G-CYC protein
was incubated with indicated concentrations of bicarbonate (NaHCO3 ) for 20 min. cGMP content
was measured as previously described. Results are expressed as means +
− S.E.M. from two
experiments. (C) Effect of pH on the cyclase activity of purified GC-G-CYC protein in the
absence or presence of NaHCO3 . The final pH of the assay buffers was adjusted to the desired
value with HCl at room temperature (25 ◦ C). The activity of the purified GC-G cyclase domain
was independent of pH when assayed in the absence (open circles) or in the presence of 30 mM
bicarbonate (filled circles). Results shown are means +
− S.E.M. from three experiments.
cellular cGMP accumulation in the GC-G-transfected neuronal
cells, which was blocked by treatment with AZ. These data
suggest that CO2 must be converted into bicarbonate for the
stimulatory effect on GC-G. Our results agree with recent
findings for GC-D showing that GC-D-positive OSNs coexpressing type II CA can sense a near-atmospheric concentration
of CO2 , possibly through the CA II-mediating conversion of
CO2 into bicarbonate for GC-D activation [9–11]. Furthermore,
c The Authors Journal compilation c 2010 Biochemical Society
272
Y.-C. Chao and others
in the aforementioned sperm processes in a bicarbonatedependent manner. However, this suggestion warrants further
investigation. Furthermore, because GC-G mRNA or protein
can be detected in several other tissues by RT–PCR (reverse
transcription–PCR) or immunohistochemistry [14,34,35], GC-G
might be involved in other bicarbonate-dependent physiological
or pathological processes. For example, elevated intracellular
bicarbonate concentrations under certain metabolic processes [36]
or with tissue ischaemia-reperfusion injury [37] may represent
putative modulating signals for GC-G activation. In agreement
with this suggestion, our recent gene-targeting study demonstrated
that GC-G may be activated and acts as an early injury
signal that promotes apoptotic and inflammatory responses in
ischaemia/reperfusion-induced acute renal injury [14].
In summary, our results reveal a novel biochemical regulatory
mechanism for an orphan GC receptor, GC-G. Given that GCG is co-expressed with the cGMP-gated ion channel CNGA3,
GC-G-positive GG neurons may transduce stimuli through an
excitatory cGMP-mediated signalling mechanism. Investigations
are required to further define the molecular regulation of GCG and unravel the precise physiological function of the GC-Gmediated cGMP signal pathway in GG neurons and in other
organs.
AUTHOR CONTRIBUTION
Figure 7
cells
CO2 activates the cyclase activity of GC-G expressed in neuronal
(A) mRNA expression of CA II in NG108 neuronal cells confirmed by RT–PCR. GAPDH was
amplified as a positive control. Omission of reverse transcriptase (−) in the RT reaction serves
as a negative control. (B) Expression of CA II protein (brown) in NG108 neuronal cells by
immunocytochemical analysis with anti-CA II antibody (lower panel). Omission of the primary
antibody serves as a control (upper panel). (C) CO2 increased the intracellular cGMP levels of
NG108 neuronal cells expressing GC-G. NG108 cells transfected with vector or GC-G expression
plasmid were cultured with or without CO2 (5 %) and with or without 1 mM AZ, an inhibitor of
CAs. *P < 0.05.
at least two downstream cGMP signalling proteins, the cGMPregulated PDE2A and the cGMP-gated ion channel CNGA3
(cyclic nucleotide-gated cation channel α-3), are expressed in both
GC-D-positive OSNs and GC-G-positive GG neurons [15,22,23].
Although CA II mRNA was not detected in GG neurons [22],
its protein expression has yet to be comprehensively examined
by immunohistochemistry, and other forms of CAs (16 different
isoenzymes [24]) may be expressed in GG. Therefore, GG cells
may respond to CO2 in a GC-G-dependent manner. However,
further studies are required to clarify this notion.
Recent studies demonstrated that neuronal cells in the GG,
a newly discovered olfactory subsystem [25–29], mediate a
chemosensory function in detecting an alarm pheromone [30] and
respond to cool temperatures [23,31,32]. Although little is known
about the identity of the alarm pheromone and the signalling
cascade involved in the chemosensory function, the cGMPgated ion channel CNGA3 was shown to play a critical role in
mediating the responses of the GG neurons to cool temperature
[23]. Thus, further investigations into whether GC-G-mediated
cGMP signalling is also functionally coupled with CNGA3 and
is involved in chemical or thermal sensation in GG is of interest.
In addition to its expression in GG neurons, GC-G protein
was shown to be highly expressed in mouse sperm [12]. An
increase in cellular bicarbonate is involved in the regulation of
sperm activities, including motility, chemotaxis, capacitation,
and the acrosomal reaction [33]. GC-G might participate
c The Authors Journal compilation c 2010 Biochemical Society
Ying-Chi Chao constructed all expression plasmids used in this study and determined
the effects of CO2 and bicarbonate on GC-G activity. Chien-Jui Cheng, Hsiu-Ting Hsieh
and Chih-Ching Lin performed the immunohistochemical analysis, cell imaging of cGMP
indicator and production of recombinant GST fusion protein respectively. Chien-Chang
Chen and Ruey-Bing Yang conceived and supervised the overall project, designed
biochemical and molecular biology experiments, and co-wrote and edited the manuscript
prior to submission.
FUNDING
This work was supported by the National Science Council [grant numbers NSC-972320-B-001-009-MY3 (to R.-B.Y.) and NSC 97-2320-B-038-018-MY3 (to C.-J.C)]. RNAi
reagents were obtained from the National RNAi Core Facility located at the Institute of
Molecular Biology/Genomic Research Center, Academia Sinica, supported by the National
Research Program for Genomic Medicine, National Science Council [grant number NSC
97-3112-B-001-016].
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Received 22 April 2010/29 July 2010; accepted 26 August 2010
Published as BJ Immediate Publication 26 August 2010, doi:10.1042/BJ20100617
c The Authors Journal compilation c 2010 Biochemical Society