Involvement of Gq/11 in signal transduction in the mammalian vomeronasal organ
Alabama State University, Biomedical Research and Training Programs, Montgomery, AL 36104-0271, USA
* Author for correspondence (e-mail: kwekesa{at}asunet.alasu.edu)
Accepted 26 November 2002
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Summary |
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Key words: pheromone, Gq/11, IP3, signal transduction, mammal, vomeronasal organ
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Introduction |
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The initial events of pheromonal detection require the activation of
specific receptors by pheromones and the transduction of the stimulus.
Molecular evidence has led to the isolation of three independent families of
vomeronasal receptor genes (VR), known as V1Rs
(Dulac and Axel, 1995), V2Rs
(Herrada and Dulac, 1997
;
Matsunami and Buck, 1997
;
Ryba and Tirindelli, 1997
) and
V3Rs (Pantages and Dulac,
2000
) that encode putative pheromone receptors. A new
classification of vomeronasal receptor genes has been proposed whereby the
V1Rs and the V3Rs are consolidated into one family
(Rodriguez et al., 2002
).
These vomeronasal receptors are unrelated to olfactory receptors. It has also
been shown that V1Rs and V3Rs are expressed in the same population of VNO
neurons, whereas V2Rs are expressed in a distinct subpopulation. These
populations are non-overlapping and there is evidence to suggest that
individual VNO neurons are likely to express only one receptor gene. Neurons
lining the apical half of the VNO neuroepithelium express V1Rs
(Dulac and Axel, 1995
),
whereas neurons in the basal half express V2Rs
(Herrada and Dulac, 1997
;
Matsunami and Buck, 1997
;
Ryba and Tirindelli, 1997
). In
mice and rats these neurons also express G-proteins in the axonal projections.
V1Rs and V3Rs express the alpha subunit of G
i2 and project
to the anterior region of the AOB, whereas V2Rs express the alpha subunit of
G
o and project to the posterior regions of the AOB
(Halpern et al., 1995
;
Wekesa and Anholt, 1999
;
Rodriguez et al., 1999
). The
expression of three types of pheromone receptors supports the idea that they
might be involved in detection of different types of chemosensory
information.
Vomeronasal chemoreception is mediated by narrowly tuned high-affinity
receptors (Leinders-Zufall et al.,
2000), and transduction involves release of
inositol-1,4,5-trisphosphate (IP3)
(Taniguchi et al., 2000
;
Wekesa and Anholt, 1997
;
Inamura et al., 1997
;
Luo et al., 1994
). In the
garter snake Thamnophis sirtalis, it has been shown that a
chemoattractant isolated from its prey induced the generation of
inositol-1,4,5-trisphosphate in the VNO
(Jiang et al., 1990
;
Luo et al., 1994
). It has been
shown that dialysis of IP3 into the turtle Geoclemys
reevesii and rat Rattus norvegicus VNO induces inward currents
(Inamura et al., 1997
;
Taniguchi et al., 1995
). We
have also previously shown that female porcine VNO can be stimulated by male
urine and seminal fluid to cause an increase of IP3
(Wekesa and Anholt, 1997
).
These results suggest that pheromonal information is mediated via the
IP3-dependent pathway in the vomeronasal receptor neurons.
Although there is some consensus that IP3 is the second
messenger in the vomeronasal system, there is no consensus as to which
G-protein(s) links receptor activation to the hydrolysis of phosphatidyl
inositol bisphosphate (PIP2). In the present study, we looked at
the effects of G-protein bacterial toxins and phospholipase C inhibitors on
responses to urine in the mouse vomeronasal organ in order to obtain
information about the signal transduction pathways. We now provide evidence in
support of signal transduction in the mammalian VNO being mediated by a member
of the Gq/11 family of G-proteins via the
IP3-dependent transduction pathway.
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Materials and Methods |
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Membrane preparations
Adult male urine was freshly collected on a daily basis and stored as
samples under argon at -80°C until used. VNOs from female mice, up to 2
weeks old, were dissected from their crevices in the nasal cavity, removed
from the cartilaginous capsule, and frozen on dry ice. The tissues were then
minced with a razor blade and subjected to sonication for 2-5 min in ice-cold
phosphate-buffered saline (PBS). The resulting suspension was layered on a 45%
(w/w) sucrose cushion and centrifuged at 4°C for 30 min at 3000 g
in a Beckman SW55Ti rotor. The membrane fraction on top of the sucrose was
collected and centrifuged as before for 15 min to pellet the membranes. The
membranes were resuspended in 100µl of ice-cold PBS. Protein was then
determined according to the method of Lowry et al.
(1951), using bovine serum
albumin (BSA) as standard.
Second messenger assays
Non-hydrolysable forms of G-protein guanosine
5'-O-(3-thiotriphosphate) (GTPS), inhibitor guanosine
5'-O-(2-thiodiphosphate) (GDPßS) and U-73122 were purchased from
Boehringer Mannheim (Indianapolis, IN, USA). Bordella Pertussis Toxin was
purchased from Calbiochem (La Jolla, CA, USA). For IP3 assays,
reactions were incubated for 1 min at 37°C in 25 mmol l-1
Tris-acetate buffer pH 7.2, 5 mmol l-1 magnesium acetate, 1 mmol
l-1 dithiothreitol (DTT), 0.5 mmol l-1 ATP, 0.1 mmol
l-1 CaCl2, 0.1 mg ml-1 BSA, 10 µmol
l-1 GTP and 20 µg VNO membrane protein. Reactions were
terminated by the addition of 1 mol l-1 trichloroacetic acid (TCA).
IP3 concentration was measured with a kit from Perkin Elmer, Inc.
(Boston, MA, USA) according to the manufacturer's instructions, based on
displacement of [3H]IP3 from a specific IP3
binding protein. In experiments using bacterial toxins, 1 µg
ml-1 pertussis (PTX) was incubated with membranes for 1 h. To
assess the effect of ADP-ribosylation by pertussis toxin on phosphoinositide
hydolysis, experiments were performed by a procedure similar to that described
by Schleifer et al. (1980
).
The toxin was activated by preincubation with 20 mmol l-1 DTT at
37°C for 15 min. The VNO membranes from prepubertal female mice at a
concentration of 0.2 mg ml-1 were exposed to 10 µg
ml-1 of the activated toxin for 1 h at 37°C in 20 mmol
l-1 Tris-HCl, pH 7.6, in the presence of 30 mmol l-1
thymidine, 1 mmol l-1 ATP, 0.1 mmol l-1 GTP, 5 mmol
l-1 MgCl2, 1 mmol l-1 EDTA, 1 mmol
l-1 DTT, 3 mmol l-1 phosphoenolpyruvate, 5 units
ml-1 pyruvate kinase, 15 µg ml-1 saponin and 0.2 mmol
l-1 NAD. Control membranes were incubated in the same reaction
mixture but in the absence of the toxin. Differences between experimental and
control animals were analyzed by analysis of variance (ANOVA).
Antibodies
Antibodies against subunits of G-proteins were obtained from
Calbiochem (La Jolla, CA, USA). The antibody against the
subunit of
Gi2 was raised against the C-terminal decapeptide KNNLDCGLF of
Gi2. The antibody against the
subunit of Go was
raised against the C-terminal peptide KNNLKECGLY of G
o. The
antibody against G
q/11 was raised against the C-terminal
peptide. Antibodies reactive with G
i2 and
G
o do not crossreact.
Western blotting
VNO membrane samples were subjected to electrophoresis on a 10%
SDS-polyacrylamide gel, followed by electrophoretic transfer onto a
nitrocellulose membrane. Strips of the membrane, containing approximately 20
µg protein, were probed with a 1000-fold dilution of normal rabbit serum or
1000-fold dilutions of rabbit antisera against specific G protein subunits
(Calbiochem, La Jolla, CA, USA). Bound antibody was visualized via a
biotinylated goat-anti-rabbit secondary antibody complexed with avidin and
biotinylated horseradish peroxidase (HRP), using Amersham's chemiluminescent
ECL detection system (Amersham, Arlington Heights, IL, USA). Migration
distances were calibrated against Kaleidoscope prestained molecular mass
markers (BioRad, Richmond, CA, USA).
Immunohistochemistry
Mice were given a lethal injection of sodium pentobarbital (50 mg
kg-1, i.p.) and perfused intracardially with PBS, followed by
extensive perfusion with 10% paraformaldehyde. The nasal cavities were
dissected and fixed overnight in 10% paraformaldehyde. Decalcification of
nasal tissue was performed for 3 days at ambient temperature using the formic
acidsodium citrate method (Luna,
1968). 5 µm thick sections through nasal tissue, formalin-fixed
and paraffin-embedded, were deparaffinized in xylene and rehydrated through
graded alcohols. The sections were pretreated with 0.1% pepsin (Sigma, St
Louis, MO, USA) in 0.01 mol l-1 HCl, pH 2.3, for 20 min to
facilitate epitope access. Following pepsin treatment, the sections were
blocked for 30 min with BEAT blocking solutions A and B (Zymed Laboratories,
San Francisco, CA, USA). They were then incubated with a 250-fold dilution of
normal rabbit serum or antiserum against G
i2,
G
o or G
q/11 in PBS, 0.05% Triton X-100
(Boehringer Mannheim, Indianapolis, IN, USA) overnight at 4°C. Following
incubation with the primary antibody, sections were washed extensively in PBS,
0.05% Triton X-100, and incubated for 10 min at room temperature with
affinity-purified biotinylated goat anti-rabbit antibody. Following a 10 min
incubation with HRP-conjugated streptavidin, antibody complexes were
visualized using 3'-amino-9'-ethylcarbazole as chromogenic
substrate. This generates a red deposit at the site of antibody binding.
Sections were counterstained with Hematoxylin, and viewed and photographed
under a Zeiss Axiophot microscope. They were then processed using Adobe
Photoshop 6.0.
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Results |
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|
|
Increase in IP3 levels induced by male urine in VNO
membranes from prepubertal female mice
To study transduction pathways activated by pheromonal stimuli from the
male, we developed a preparation enriched in microvillar membranes from VNOs
of prepubertal female mice. Incubation of microvillar membranes with male
urine results in a robust increase in IP3 as compared to the
control (P<0.05). This response is mimicked by GTPS and
blocked by GDPßS (Fig. 3).
We therefore concluded that female VNO membranes respond to stimuli in male
urine with an increase in IP3 via a G-protein-coupled
pathway. In order to determine whether the production of IP3 was
mediated by phospholipase C, we incubated our membranes with a highly
selective, cell-permeable inhibitor of PLC (U-73122, 10 µmol
l-1). We observed that incubation of VNO membranes with a PLC
inhibitor blocked the production of IP3 (P<0.05). In
order to determine which G-protein mediated the activation of PLC, we
incubated our VNO membranes in pertussis toxin before stimulation. We observed
that incubation of VNO membranes with PTX in the presence of male urine still
resulted in an increase of IP3 levels. This response was not
different from that of the control membranes, which were only stimulated with
male urine in the absence of PTX. We concluded that pheromonal stimulation of
VNO membranes results in an increase of IP3 levels via
activation of PLC by the alpha subunit of the Gq/11 class of
G-proteins.
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Discussion |
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In the present study we looked at the effects of phospholipase C inhibitors
and G protein toxins in the signal transduction pathways. By use of bacterial
toxins, which are commonly used to classify G proteins, we were able to show
that VNO microvilli membranes incubated with pertussis toxin (PTX) are capable
of being stimulated by urine to produce IP3. Members of the
Gq family, e.g. G
11 and
G
q, lack a recognition site for ribosylation by PTX
(Pang and Sternweis, 1990
;
Strathmann and Simon, 1990
;
Blank et al., 1991
;
Sternweis et al., 1992
)
whereas G
i and G
o have a recognition site
and undergo ADP ribosylation in the presence of PTX. Since we do not see any
changes in production of IP3 in the presence of PTX, we conclude
that the main G-protein involved in the production of IP3 is
PTX-insensitive, such as the G
q/11 class of G-proteins.
The presence of G-protein, recognized by anti-Gq on
western blots of VNO microvillar membrane preparations, indicates that a
substrate is present to explain the physiological effect of this antibody and
is consistent with the possibility suggested by the ribosylation experiments
that the G-proteins are members of the G
q family. These
results are consistent with the observation that G
q
activates PLC and causes an increase in IP3 in our membrane
preparations. The ability to block this response with a PLC inhibitor such as
U-73122 (Bleasdale et al.,
1990
; Inamura et al.,
1997
) further confirms the G
q mediated pathway.
It has also been shown that when U-73122 is applied to the neuroepithelium it
blocks spiking in response to urine (Holy
et al., 2000
). G
q and its close relatives
activate the ß1, ß2 and ß3-isoforms of PLC and, in contrast to
G
i2 and G
o, are not inhibited by pertussis
toxin (Taylor et al., 1991
;
Lee et al., 1992
). In addition
to the
subunits of G proteins, ß
subunits can in some
instances mediate inhibition or activation of adenylate cyclase
(Tang and Gilman, 1991
;
Clapham and Neer, 1993
) or
stimulation of phospholipase C, especially the ß2-isoform
(Camps et al., 1992
;
Boyer et al., 1992
;
Katz et al., 1992
;
Clapham and Neer, 1993
) and we
cannot exclude a role for the ß
subunits in mediating vomeronasal
signal transduction. However, our experiments are designed primarily to
determine the relative contributions of the distinct
subunits of
Gi2, Go and Gq/11 to activation of
phospholipase C in the VNO.
Pheromonal-induced increases in IP3 levels imply a role for
calcium in vomeronasal signal transduction. The phosphoinositide cycle
responds to the actions of an agonist at the receptor level by hydrolysis of
PIP2, resulting in the generation of IP3 and
diacylglycerol (DAG) (Neer and Clapham,
1988). DAG enhances the activity of protein kinase C (PKC) by
rendering it more sensitive to stimulation by calcium
(Nishizuka, 1988
), while
IP3 stimulates the release of calcium from the endoplasmic
reticulum stores (Berridge,
1987
; Berridge and Irvine,
1989
). Previous experiments by
Leinders-Zufall et al., 2000
)
using slice preparations showed that pheromone application to VN results in a
robust and reproducible increase in calcium levels. These results are
consistent with other studies showing that urine stimulation activates a
calcium-permeable cation-selective inward current in rat vomeronasal neurons
(Inamura and Kashiwayanagi,
2000
). A role for IP3 signaling pathways is also
suggested by the presence of the transient receptor potential (TRP) channel
family, TRP2 in the VNO (Liman et al.,
1999
). This supports the idea that the increases in IP3
levels are G
q related. In Drosophila it has been
shown that during the photoisomerization process, rhodopsin, a Gq
class molecule, is activated, which triggers an IP3 signaling
cascade and leads to the opening of cation-sensitive channels dTRP and dTRPL.
It is possible that pheromonal transduction might act in a similar manner.
Although it is unclear how TRP2 is activated, members of the TRP channel
family are often coupled to PLC activation and can be gated by products of the
PIP2 second messenger cascade. Consistent with these findings, VNO
responses to components of urine have been blocked by pharmacological
inhibitors of PLC (Holy et al.,
2000
). Therefore it possible that activation of pheromone
receptors by G
q/11 leads to the stimulation of PLC.
Elevation of IP3 or DAG would then lead to the activation of TRP2
channels and membrane depolarization.
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Acknowledgments |
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