Selective Activation of G Protein Subtypes in the Vomeronasal
Organ upon Stimulation with Urine-derived Compounds*
Jürgen
Krieger
,
Annette
Schmitt
,
Diedrich
Löbel
,
Thomas
Gudermann§,
Günter
Schultz§,
Heinz
Breer
, and
Ingrid
Boekhoff
¶
From the
Universität Stuttgart-Hohenheim,
Institut für Physiologie, Garbenstr. 30, 70593 Stuttgart,
Germany and the § Freie Universität Berlin,
Institut für Pharmakologie, Thielallee 69-73,
14195 Berlin, Germany
 |
ABSTRACT |
Chemosensory neurons in the vomeronasal organ
(VNO) detect pheromones related to social and reproductive behavior in
most terrestrial vertebrates. Current evidence indicate that the
chemoelectrical transduction process is mediated by G protein-coupled
second messenger cascades. In the present study, attempts were made to
identify the G protein subtypes which are activated upon stimulation
with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol
1,3,4-trisphosphate formation induced by urinary
stimuli and to immunoprecipitate G
-subunits, activation dependently
labeled with [
-32P]GTP azidoanilide. The results of
both experimental approaches indicate that stimulation of female VNO
membrane preparations with male urine samples induces activation of
Gi as well as Go subtypes. Experiments using
different fractions of urine revealed that upon stimulation with
lipophilic volatile odorants, only Gi proteins were
activated, whereas Go activation was elicited by
2u-globulin, a major urinary protein, which is a member
of the lipocalin superfamily. Since each G protein subtype is
stereotypically coexpressed with one of the two structurally different
candidate pheromone receptors (V1R and V2R), the results provide the
first experimental evidence that V1Rs coexpressed with Gi
may be activated by lipophilic probably volatile odorants, whereas V2Rs
coexpressed with Go seem to be specialized to interact with
pheromonal components of proteinaceous nature.
 |
INTRODUCTION |
Terrestrial vertebrates detect chemical signals via sensory
neurons located in two anatomically distinct systems: the nasal olfactory epithelium (OE)1
and the vomeronasal organ (VNO). Whereas the main olfactory system is
responsible for the "conventional" sense of smell, the VNO appears
to specifically detect pheromones, thereby inducing a distinct social
or sexual behavior or endocrine response (1-3). In rodents, the major
source of phermones seems to be the urine, however, only few volatile
(4) and non-volatile (5) urinary substances producing a definite
endocrine or behavioral response have been identified. The best
characterized non-volatile urinary components in male mouse urine are
the "major urinary proteins" (6). Major urinary proteins have been
suggested to be involved in puberty acceleration (5, 7, 8), an
endocrinological effect prevented by lesions of the VNO or the
accessory olfactory bulb (1). Major urinary proteins of mice and the
rat equivalent
2u-globulin (9) belong to the superfamily
of lipocalins, a structurally homologous but diverse family of
extracellular proteins, characterized by their ability to bind small,
principally hydrophobic molecules (10). Although recent structural
studies have characterized the naturally bound volatile substances
(11), major urinary proteins lacking the natural volatiles remain
active (8) suggesting that proteins themselves act as pheromones.
The molecular mechanisms responsible for the detection of pheromones
and the signal transduction processes in the VNO are not well
understood. Initially, it has been suggested that the transduction
mechanisms in the VNO would resemble those in the olfactory epithelium,
but some of the specific molecular components involved in the
transduction of odorants in receptor neurons of the OE, like
Golf, adenylyl cylase (AC) subtype III, and the
-subunit of the cyclic nucleotide-gated cation channel could not be detected in
the neuroepithelium of the VNO (12, 13). Moreover, recent molecular
biology advances implicate that neurons in the VNO utilize another set
of receptor genes than the olfactory epithelium. Two novel and
structurally unrelated multigene families of putative pheromone
receptors have been identified (V1R and V2R) which are exclusively
expressed in neurons of the VNO (14-17). Although receptors of both
families represent members of G protein-coupled receptors, the
structural differences especially in the N-terminal region led to the
concept that each receptor family may be tuned to recognize a distinct
class of ligands.
Receptors of each family show a distinct nonoverlapping expression
pattern within two major laminar zones. Whereas V1Rs are expressed in
the apical half of the neuroepithelium, V2Rs are restricted to the
basal region of the epithelium. An identical spatial expression pattern
has also been observed for two types of G protein
-subunits. Whereas
Gi is expressed only in apical neurons, Go
expression is restricted to neurons in the basal half of the VNO
neuroepithelium (13, 14, 18). The stereotypical distribution of both
receptor families with distinct G protein subtypes implies a linkage
between both components.
The present study was aimed to characterize the G protein subtypes in
the rat vomeronasal organ activated upon stimulation with male urine by
performing photoaffinity labeling experiments. Furthermore, antibodies
for different G protein subtypes were employed to interfere with the
urine-induced IP3 signaling. To evaluate whether there is
any correlation between the chemical properties of applied urinary
stimuli and the activation of distinct G protein subtypes, stimulation
experiments were performed with different fractions of urine as
well as with purified
2u-globulin.
 |
EXPERIMENTAL PROCEDURES |
Materials
Male and female adult Sprague-Dawley rats were purchased from
Charles River, Sulzfeld, Germany. The odorant menthone was provided by
DROM (Baierbrunn, Germany), amyl acetate was purchased from Sigma
(Deisenhofen, Germany). Hydroxyapatite Type I was obtained from Bio-Rad
(München, Germany), the Centricon concentrators were purchased
from Millipore (Eschborn, Germany), enterokinase was from Boehringer
(Mannheim, Germany). The radioligand assay kits for cAMP (adenosine
3',5'-cyclic phosphate) and myo-[3H]inositol
1,4,5-trisphosphate determination were provided by Amersham;
[
-32P]GTP was purchased from NEN Life Science Products
Inc. Sources of other materials have been described (19).
Methods
Antisera--
Antisera against G protein subunits were obtained
after injection of synthetic peptides representing subtype-specific
regions of different subunits into rabbits as described previously (20) or provided by Santa Cruz Biotechnology, Santa Cruz, CA. The peptide sequences used to raise the antisera are shown in Table I.
Urine Collection and Purification--
Urine from male fertile
rats (12-14 weeks old) was collected daily, pooled, centrifuged to
remove cells (5 min, 5, 500 × g), and stored as
aliquots at
70 °C until use. To extract hydrophobic volatile
odorants, a 2-ml volume of pooled male urine was treated with 2 ml of
dichloromethane; following separation of the organic and water phase by
centrifugation (10 min, 6,000 × g), both phases were
collected and stored at
70 °C.
Purification of
2u-Globulin--
Urine was collected
from fertile male rats, pooled, and stored immediately at
70 °C.
After thawing to 4 °C, a 50-ml pool was centrifuged at 5,000 × g for 30 min at 4 °C, and the clear urine was dialyzed
against 4 mM sodium phosphate buffer, pH 6.8. The
purification of
2u-globulin was performed according to
Holmquist et al. (21). Briefly, after addition of
phenylmethylsulfonyl fluoride to a final concentration of 0.1 mM, the samples were applied to a 1.5 × 10-cm
hydroxyapatite column with a flow rate of 0.5 ml/min. The column was
washed with 10 times column volumes of 4 mM sodium
phosphate buffer, pH 6.8. Elution was performed using a linear 4-400
mM sodium phosphate buffer gradient, pH 8.0. Total protein
was evaluated by monitoring the A280,
corresponding fractions were pooled and analyzed by SDS-PAGE. Fractions
containing the 18-kDa proteins were collected, dialyzed against
reaction buffer (200 mM NaCl, 10 mM EGTA, 50 mM Mops, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.05% sodium cholate, 1 mM
ATP, and 4 µM GTP, 12 nM CaCl2,
pH 7.4) and concentrated using Centricon-10 to a final protein
concentration of 17 mg/ml.
Construction of Expression Plasmids--
DNA-related methods
were performed according to standard procedures (22). The coding
sequences of
2u-globulins were amplified by polymerase
chain reaction using cDNA obtained by reverse transcription of
poly(A)+ RNA from male rat liver. A recombinant
2u-globulin with an N-terminal sequence corresponding to
the endogenous protein was generated by using an extended
5'-oligonucleotide in the polymerase chain reaction that contained a
BamHI site (underlined) and the sequence coding for an
enterokinase cleavage site in-frame to the coding sequence:
2u-forward
5'-ggatccgtacgacgatgacgataaggaagaagctagttccacaagagg-3'; as
an antisense primer oligonucleotide containing an HindIII
site (underlined), the following oligonucleotide was used:
2u-reverse 5'-aaagcttcgtcaac-ctcaggcctggagacagcg-3'. The
amplification products were cloned into the pGEM-T vector. All derived
plasmid constructs were confirmed by DNA sequencing. For subcloning
into the expression vector pTrcHis, the
2u-globulin
sequences from pGEM-T-plasmids were cut out with BamHI and
HindIII restriction enzymes and ligated in-frame into the
corresponding vector sites. The expression constructs were transformed
into Escherichia coli BL21 (DE3).
Growth of Cells and Protein Purification--
100 ml of
LB-medium with ampicillin (100 µg/ml) was inoculated with an
overnight culture in the same medium and grown at 37 °C. Protein
expression was induced at A600 of 0.5-0.7 with
0.2 mM isopropyl-
-D-thiogalactoside, and
growth was continued for 3 h. Cells were harvested by
centrifugation at 2,000 × g for 15 min and resuspended
in 20 ml of 50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.25 mg/ml lysozyme,
and 1 mM phenylmethylsulfonyl fluoride and stored on ice
for 1 h. For damage of spheroplastes, cells were frozen in liquid
nitrogen and thawed at room temperature. After sonification with a tip sonicator for 2 min on ice, the soluble material was separated from
cell debris and inclusion bodies by centrifugation at 30,000 × g for 30 min. The supernatant was collected and used for
affinity chromatography.
For purification of His-tagged proteins, the cytosol was loaded onto a
Ni-NTA agarose column equilibrated against washing buffer (50 mM sodium phosphate buffer, pH 8.0, 300 mM
NaCl, and 20 mM imidazole) and washed with 5 column volumes
of washing buffer. Subsequently, bound protein was eluted as single
peak by increasing the concentration of imidazole to 250 mM. Fractions of 1 ml were collected and protein
concentrations were monitored by A280;
corresponding fractions were pooled and dialyzed twice for 8 h
against 50 mM Tris/HCl, pH 7.5.
Protein concentration was adjusted to 1 mg/ml with 50 mM
Tris/HCl, pH 7.5, 0.1% Tween 20. Cleavage of fusion protein was
performed by incubation with 1/40 (w/w) enterokinase for 16 h at
37 °C. Subsequently, the NaCl concentration was increased to a final concentration of 500 mM, and agarose-coupled soybean
trypsin inhibitor was added; after an incubation for 2 h at
4 °C, soybean trypsin inhibitor bound enterokinase was precipitated
by centrifugation (2,000 × g for 10 min) and the
supernatant was recovered.
Preparation of Vomeronasal Organ Membranes--
Membrane
fractions of the VNO were prepared as described previously (23).
Briefly, VNOs removed from fertile male rats were washed twice in
Ringer's solution (120 mM NaCl, 5 mM KCl, 1.6 mM K2HPO, 25 mM NaHCO3,
7.5 mM glucose, pH 7.4) to remove superficial blood and
debris and subsequently frozen in liqiud nitrogen. Each pooled sample
(30-60 animals) was thawed on ice, minced, and subsequently subjected
to Ringer's solution containing 10 mM calcium chloride; after gently stirring for 10 min at 4 °C, debris was removed by centrifugation (10 min, 3,000 × g), the resulting
supernatant was collected and the pellet was resuspended again in
Ringer's solution containing CaCl2 as described above. The
pooled supernatants containing the membrane fractions were centrifuged
again for 30 min at 48,000 × g and the resulting
pellet was resuspended in aliquots (100-500 µg in 500 µl) in
hypotonic buffer (10 mM Tris/HCl, 3 mM
MgCl2, 2 mM EGTA, pH 7.4) and stored at
70 °C. Olfactory cilia of the OE were obtained using the
calcium-shock method as described previously (24).
Stimulation Experiments and Second Messenger
Determination--
Stimulation experiments, performed with male rat
urine, separated fractions of urine, isolated
2u-globulin of male urine, and recombinant
2u-globulin were performed as described previously (24).
Briefly, 300 µl of reaction buffer (200 mM NaCl, 10 mM EGTA, 50 mM Mops, 2.5 mM
MgCl2, 1 mM dithiothreitol, 0.05% sodium
cholate, 1 mM ATP, and 4 µM GTP, pH 7.4)
including 12 nM free calcium calculated and adjusted as
described (25), was prewarmed at 37 °C with or without odorants. The
reaction was started by the addition of 60 µl of VNO membrane
preparations, incubated for 2 min at 37 °C and stopped by 7%
ice-cold perchloric acid (200 µl) before the concentration of
IP3 was determined according to Palmer et al.
(26). To prevent degradation of IP3, stimulation
experiments were performed in the presence of 10 mM LiCl
(final concentration during incubation). To determine the influence of
the subtype-specific G protein
-subunit antisera on the efficiency
of urine-induced second messenger responses, membrane preparations of
the VNO were preincubated with the indicated dilutions of the specific
antisera and subsequently stimulated with urine or purified fractions
of urine.
Photolabeling of Activated G
Proteins--
[
-32P]GTP azidoanilide was synthesized
and purified as described (27); receptor-dependent G
protein labeling was performed as described previously (20). Briefly,
frozen VNO membrane preparations in hypotonic buffer were centrifuged
(10 min, 12,000 × g, 4 °C) and resuspended in
double concentrated labeling buffer (60 mM HEPES, 5 mM MgCl2, 200 mM NaCl, 200 µM EDTA, 500 µM GDP, pH 7.4). 10 µl of
urine, general odorants, separated urine fractions (diluted in water),
or
2u-globulin (diluted in reaction buffer) were adapted to 37 °C with 20 µl of [
-32-P]GTP azidoanilide
(4 × 106 cpm/tube). The reaction was started by
adding 30 µl of VNO membrane preparations (1-2 µg/µl); after an
incubation period of 2 min at 37 °C the reaction was terminated by
cooling the samples to 4 °C. Excess [
-32P]GTP
azidoanilide was removed by centrifugation (5 min, 12,000 × g, 4 °C). The pellet was resuspended in labeling buffer
containing 2 mM dithiothreitol, placed on a Parafilm-coated
metal plate (4 °C), and irradiated for 30 s with a 254-nm UV
lamp (150 W, Vl-100 Grid-Tube, Herolab GmbH).
Immunoprecipitation--
Photolabeled membranes were pelleted
and solubilized in 2% (w/v) SDS for 10 min at room temperature before
20 µl/tube of precipitation buffer (10 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1%
desoxycholate, 1% Tergitol Nonidet P-40, 10 µg/ml aprotinin, pH 7.4)
and 5 µl/tube of the indicated, undiluted subtype-specific G protein
-subunit antisera were added.
After 1 h at 4 °C under constant rotation, 60 µl of Protein
A-Sepharose (3 mg) was added and the samples were incubated overnight at 4 °C. Thereafter the Sepharose beads were pelleted (1 min, 12,000 × g, 4 °C) and washed twice with 1 ml of
washing buffer A (50 mM Tris/HCl, 600 mM NaCl,
0.5% SDS, 1% Tergitol Nonidet P-40, pH 7.4) and twice with washing
buffer B (100 mM Tris/HCl, 300 mM NaCl, 10 mM EDTA, pH 7.4). Preparation of the samples for SDS-PAGE
was performed as described Laugwitz et al. (20).
Incorporated [
-32P]GTP azidoanilide was determined
densitometrically after gel-exposure to a PhosphorImager (Fuji).
 |
RESULTS |
Based on a recent study indicating that treatment of vomeronasal
organ slice preparations of rats with urine dilutions elicit an
increased impulse frequency of sensory neurons which can be blocked by
phospholipase C inhibitors (28), we set out to explore whether
urine-induced IP3 responses in membrane preparations of VNOs from female rat are mediated by a G protein-controlled reaction cascade. IP3 responses induced upon stimulation with
different concentrations of male urine (0.1 to 4%, v/v) were
determined in the presence of GTP or GDP. The results in Fig.
1 demonstrate that male urine elicited a
dose-dependent increase in the formation of
IP3; this response was only observed in the presence of GTP indicating that urine-induced phospholipase C activation in the VNO is
mediated via G proteins.

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Fig. 1.
GTP dependence of the production of
IP3 induced upon stimulation with different dilutions of
male rat urine. Membrane preparations of VNOs of female rats were
stimulated with different dilutions of male rat urine ranging from 0.1 to 4% (v/v), either in the presence of 4 µM GTP or
alternatively in the presence of 10 µM GDP. Data are
calculated as urine-induced IP3 formation. Basal level of
IP3 was 33.9 ± 12.9 pmol/mg of protein. Values are
the means of three independent experiments ± S.D.
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|
Immunohistochemical studies (18, 29, 30) and in situ
hybridization experiments (13) have demonstrated that different G
proteins
-subunits (Gs, Gi2, Go,
and Gq/11/13) are expressed in neurons of the VNO. Toward
an identification of distinct G protein subtypes which are activated
upon stimulation with urine, a photoaffinity labeling approach was
employed using the photoreactive, hydrolysis resistant
[
-32P]GTP azidoanilide (27, 20). Female VNO membrane
preparations were stimulated with two different concentrations of male
urine in the presence of [
-32P]GTP azidoanilide, and
subsequently distinct G protein
-subunits were immunoprecipitated
(24) using subtype-specific antibodies for different G protein
-subtypes (Table I).
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Table I
Peptide sequences used for generation of antibodies
(C) indicates that a N-terminal cysteine was added to the original
peptide sequence in order to facilitate coupling to keyhold limpet
hemocyanin.
|
|
Fig. 2 shows that urine addition
significantly enhanced incorporation of [
-32P]GTP
azidoanilide into G
i as well as G
o
subtypes. Quantification of the labeling intensity indicates that
stimulation with male urine (0.2%, v/v) induced a 57% increase in
G
i labeling and a nearly 100% increase in the
[
-32P]GTP azidoanilide incorporation into
G
o compared with control samples. In contrast, proteins
precipitated with an antiserum specific for G
s and
G
olf (AS 348) as well as G
q subtypes (AS 368) show only very weak incorporation of [
-32P]GTP
azidoanilide under control conditions, and no increase upon urine
stimulation was registered (data not shown).

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Fig. 2.
Effect of different concentrations of male
rat urine on photoaffinity labeling of
G i and
G o. Vomeronasal organ
preparations of female rat were photolabeled in the absence (control)
or presence of two different concentrations of male rat urine indicated
in percent v/v. Labeled membranes were solubilized, and
immunoprecipitations were performed using either the antibody C-10,
specific for G i (top panel), or the selective
G o antibody K-20 (bottom panel). The
autoradiograms, showing the 40-kDa regions of a SDS-PAGE, are
representative of three independent experiments each giving very
similar results. The intensity of labeling, represented as
photostimulated luminescence (PSL), was determined
densitometrically. Data were calculated as percentage of the basal
photostimulated luminescence obtained in the absence of urine.
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Antibodies for distinct G protein
-subtypes have been successfully
used to selectively inhibit ligand-induced phosphatidylinositol bisphosphate hydrolysis (24, 31). Accordingly, membrane preparations of
female VNOs from rat were pretreated with different concentrations of
subtype-specific antibodies and subsequently stimulated with a 2%
(v/v) dilution of male urine. As demonstrated in Fig.
3A, preincubation with the
G
i common antibody C-10 or with the
G
o-specific antibody K-20 attenuated the urine-induced
IP3 response in a concentration-dependent manner; 50% inhibition was obtained at a 1:500 dilution of either antibody. In contrast, antibody AS 348 specific for G
s
subtypes as well as AS 368 selective for the G
q
subfamily did not affect the responsiveness significantly (Fig.
3B). These results indicate that the IP3
response in female VNO preparations induced upon stimulation with a
complex pheromonal mixture (male urine) is mediated via Gi
as well as Go proteins.

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Fig. 3.
Concentration-dependent
inhibition of urine-induced IP3 formation by different
G subtype-specific antibodies. Aliquots
of membrane preparations of female VNOs were pretreated with different
concentrations of G subtype-specific antibodies (A, C-10
for G i and K-20 for G o; B,
AS-368 for G q and AS-348 for G s) and
subsequently samples were stimulated with a 2% (v/v) dilution of male
rat urine. The basal level of IP3 in the VNO preparations
under control conditions (38.1 ± 6.8 pmol/mg of protein) was not
affected upon pretreatment with the different G protein antibodies. The
data are calculated as percent of urine-induced IP3
formation (57.9 ± 13.0 pmol/mg of protein). Values are the means
of three experiments ± S.D.
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Functional and chemical analysis of urine from rodents have shown that
pheromonal components are found in both the volatile as well as
non-volatile fraction (5, 32). The volatile components can be extracted
from whole male urine using dichloromethane (8). Using this procedure,
the dichloromethane-extractable urinary components as well as the
remaining aqueous fraction were subsequently analyzed for their ability
to induce IP3 responses in VNO membrane preparations. As
shown in Fig. 4, compounds
extracted with dichloromethane induced a
concentration-dependent increase in the level of
IP3, indicating that this fraction contained active
ligands; in contrast, the aqueous fraction did not induce any changes
in the concentration of IP3 compared with control samples
(Fig. 4). Although it is unclear why the aqueous fraction is inactive
it is conceivable that the proteins may be denaturated by
dichloromethane.

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Fig. 4.
Dose-response curve of IP3
formation induced upon stimulation with urinary fractions.
Different concentrations of organic urinary substances of male rat
urine extracted with dichloromethane or the remaining aqueous urinary
components were applied to VNO membrane preparations. Basal levels of
IP3 obtained upon incubating of VNO preparations only with
reaction buffer were 24.03 ± 7.3 pmol/mg of protein; treatment of
samples with a 5% (v/v) dilution of pure dichloromethane did not
affect the concentration of IP3 (26.0 ± 4.8 pmol/mg
of protein). Data are presented as ligand-induced IP3
concentration in picomole/mg of protein and are the mean values of
three experiments ± S.D.
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In order to address the issue of which G protein subtypes are labeled
upon stimulation with the volatile urinary components, additional
photoaffinity labeling experiments were performed employing different
concentrations of the dichloromethane extract (0.02-0.4%, v/v). As
demonstrated in Fig. 5, the organic
extract induced a dose-dependent incorporation of
[
-32P]GTP azidoanilide into G
i, whereas
a stimulus-induced labeling of G
o was not detectable
(Fig. 5). These results indicate that Gi proteins but not
Go proteins are activated upon stimulation with volatile
urinary components.

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Fig. 5.
Effect of increasing concentrations of an
organic extract of male rat urine on photolabeling of
G i and
G o. Membrane preparations of
VNOs of female rats were stimulated with different concentrations of a
dichloromethane extract of male rat urine ranging from 0.0 to 0.4%
(v/v) (indicated as numbers) in the presence of
[ -32P]GTP azidoanilide. G i was
precipitated with the antibody AS 266 (top panel), whereas
G o was precipitated with the antibody K-20 (bottom
panel). The autoradiograms showing the 40-kDa region of a
SDS-PAGE are representative of three independent experiments with
similar results. Quantification of incorporated
[ -32P]GTP azidoanilide was determined by densitometric
analysis of autoradiograms presented as photostimulated luminescence
(PSL) and calculated as percentage of the basal
photolabeling obtained in the presence of 0.4% (v/v)
dichloromethane.
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To confirm this observation, the effects of G
i as well
as G
o antibodies on dichloromethane extract-induced
IP3 signaling were analyzed. VNO preparations were
pretreated with different concentrations of each of the two
subtype-specific antibodies and subsequently stimulated with a 2%
(v/v) dilution of the organic urinary extract. Whereas the
dichloromethane extract induced IP3 formation was only
slightly affected upon pretreatment with high concentrations of the
G
o antibody (1:250 dilution), G
i
antibodies caused already a strong inhibition at very low antibody
concentrations: at a 1:2000 dilution of the G
i antibody,
the dichloromethane extract-induced IP3 response was
blocked by 70% (Fig. 6).

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Fig. 6.
Effect of subtype-specific antibodies for
different G proteins on IP3 formation induced upon
stimulation with an organic urinary extract. Membrane preparations
of female rat VNOs were incubated with different concentrations of
G i (C-10) as well as G o (K-20) specific
antibodies and subsequently stimulated with a 2% (v/v) dilution of the
urinary dichloromethane extract. Data are calculated as percent of
ligand-induced IP3 formation (54.2 ± 8.6 pmol/mg of
protein); the basal level of IP3 was 32.2 ± 13.0 pmol/mg of protein. Data are the means of three independent
experiments ± S.D.
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Previous electrophysiological experiments have demonstrated that the
vertebrate VNO system is not only selective to a narrow range of
pheromonal components, but in addition is sensitive to various general
odorants normally known to be reorganized by the olfactory epithelium
(33). In order to address the question which G protein subtypes are
activated upon stimulation with volatile odorants, amyl acetate and
menthone, which elicit electrical responses in turtle VNO neurons (33),
were applied in photolabeling experiments. Membrane preparations of rat
VNOs were stimulated with different concentrations of the two general
odorants and subsequently G
i as well as
G
o subtypes were precipitated by means of
subtype-specific antibodies. Application of menthone (Fig.
7) as well as amyl acetate (not shown)
induced a concentration-dependent labeling of
G
i, however, neither menthone (Fig. 7) nor amyl acetate
(not shown) induced a significant incorporation of the GTP analog into
G
o.

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Fig. 7.
Effect of menthone on photolabeling of
G i and
G o. Membrane preparations of
female rat VNOs were stimulated with increasing concentrations of the
minty odorant menthone (1-100 µM), and subsequently
G i (top panel) as well as G o
(bottom panel) were immunoprecipitated with subtype-specific
antibodies (C-10 for G i; K-20 for G o).
The autoradiograms show the 40-kDa region of a SDS-PAGE and are
representative of three independent experiments with similar results.
The intensity of the photolabeling of G i as well as
G o was evaluated densitometrically, data were calculated
as percentage of the basal photolabeling obtained in the absence of
odorant.
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The observation that neither organic components from male urine nor
general volatile odorants induced Go activation led to the
idea that urinary proteins may be ligand candidates for Go stimulation. In urine of fertile male rats,
androgen-dependent
2u-globulin, a member of
the lipocalin superfamily (10), is the quantitatively major protein
(9). Therefore,
2u-globulin was purified from male urine
as described by Holmquist et al. (21) and assessed for its
capability to induce IP3 formation. The results of these
experiments (Fig. 8) indicate that
purified
2u-globulin induced a very strong
concentration-dependent increase in the concentration of
IP3.

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Fig. 8.
Tissue specificity of the production of
IP3 by
2µ-globulin in
female VNO preparations. A, concentration-response
curves of 2u-globulin-induced IP3 formation.
Membrane preparations of female rat VNOs were stimulated with different
concentrations of either 2u-globulin purified from male
rat urine or recombinant protein expressed in E. coli. Data
are calculated as ligand-induced IP3 formation; the basal
level in the absence of the lipocalin was 23.6 ± 8.8 pmol/mg of
protein. Data are the means of three independent experiments ± S.D. B, effect of recombinant 2u-globulin on
IP3 formation in olfactory cilia of the OE. Isolated
olfactory cilia were incubated either with reaction buffer
(control) or stimulated with recombinant
2u-globulin (50 µM), respectively, the
nonhydrolyzable GTP analogue GTP S (1.6 µM), and
subsequently the concentration of IP3 was determined.
|
|
Since the procedure for isolating native
2u-globulin,
such as gel permeation and dialysis purification steps, are not
sufficient to separate noncovalently bound volatile urinary compounds
(34), recombinant
2u-globulin, heterologously expressed
in E. coli, was employed. As shown in Fig. 8A,
application of recombinant
2u-globulin also induced an
elevated IP3 response; however, the efficacy was
considerably smaller compared with native purified urinary protein.
Nevertheless, the
2u-globulin-induced second messenger
response is specific since stimulation of olfactory cilia from the OE
did not show any changes in IP3 concentration (Fig.
8B). To characterize the G protein subtype involved in
recombinant
2u-globulin-induced IP3
signaling, the effect of subtype-specific antibodies on IP3
formation was analyzed. As documented in Fig. 9, G
i antibodies did not
alter the responsiveness to
2u-globulin, even at rather
high concentrations. In contrast, antibodies against G
o
significantly attenuate IP3 formation in a
concentration-dependent manner. This observation was confirmed
in photoaffinity labeling experiments; stimulating VNO membrane
preparations with 1 µM recombinant
2u-globulin led to a significant incorporation of
[
-32P]GTP azidoanilide into G
o, whereas
labeling of G
i subtypes was not changed (Fig.
10).

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|
Fig. 9.
Selective blockade of
2u-globulin induced IP3
formation by G o-specific
antibodies. Membrane preparations of female rat VNOs were
pretreated for 10 min on ice with different concentrations of
G i (C-10) or G o (K-20)-specific
antibodies and subsequently stimulated with 50 µM
recombinant 2u-globulin. Data are calculated as % of
ligand-induced IP3 formation (51.2 ± 5.0 pmol/mg of
protein); the basal level of IP3 in the absence of the
lipocalin was 31.7 ± 4.7 pmol/mg of protein. Data are the means
of three independent experiments ± S.D.
|
|

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|
Fig. 10.
Photoaffinity labeling of
G i and
G o subtypes upon stimulation with
recombinant 2u-globulin. VNO
membrane preparations from female rats were stimulated with 1 µM recombinant 2u-globulin; and
subsequently G o (top panel) as well as
G i (bottom panel) were precipitated with
subtype-specific antibodies (G i, C-10;
G o, K-20). The autoradiograms, showing the 40-kDa region
of a SDS-PAGE, are representative of three independent experiments with
similar results.
|
|
 |
DISCUSSION |
In the present study, G protein subtypes involved in urine-induced
IP3 formation in VNO preparations were characterized by two
different experimental approaches. Subtype-specific G protein antibodies have been used as tools to selectively inhibit urine-induced IP3 responses and to immunoprecipitate G protein subtypes
photoaffinity labeled with the hydrolysis resistant GTP analogue
[
-32P]GTP azidoanilide. The results of both
experimental approaches indicate that whole male urine induces
activation of Gi as well as Go subtypes,
suggesting a role for both G protein subtypes in transducing pheromonal
responses mediated by urinary components. Activation of Gi
and Go subtypes in the VNO appears to be realized by two
structurally different classes of odorants. Whereas Gi activation was only observed upon stimulation with lipophilic, possibly
volatile components, Go activation was elicited by
2u-globulin, a major urinary protein, which is a member
of the lipocalin superfamily. Previous studies have shown that
lipocalin proteins, like
2u-globulin and mouse urinary
proteins, share the ability to bind volatile pheromones (34),
suggesting that both proteins act as carrier proteins for hydrophobic
ligands; however, the results of the present study suggest that
2u-globulin is not merely a carrier of volatile
pheromones but is able to induce receptor-mediated Go
activation. This observation is in line with previous behavioral studies indicating that members of the lipocalin superfamily may by
themselves be active in VNO-mediated pheromonal responses. It has been
demonstrated that mouse urinary proteins separated from volatile
ligands, either by extraction or competitive displacement, were still
active in bioassays (8). Furthermore, recombinant aphrodisin, a
lipocalin-like protein from the female golden hamster vaginal
discharge, expressed in E. coli induced a specific
copulatory response in male golden hamster (35). This observation
indicates that lipocalins can act as phermones by themselves. However,
a functional role for small ligands cannot be excluded, since
recombinant aphrodisin only gained its full activity when it was
supplemented with organic extracts of the vaginal discharge (35),
suggesting that ligands may modulate the activity of lipocalins by
altering its conformation.
A main result of the present study is the observation that both G
protein subtypes, activated by different classes of urinary stimuli,
led to the activation of phospholipase C and the generation of
IP3. Thus, it is the question whether or not there is a
physiological significance of having two populations of VNO neurons
with distinct activated G protein subtypes that activate the same
downstream effector. Activation of phospholipase C by pertussis
toxin-sensitive G protein subtypes, like Gi and
Go, appears to be mediated by 
-subunits of the
trimeric G protein (36). Therefore one might speculate whether the
simultaneously released
-subunits of Gi and
Go may affect pathways not monitored in this study,
suggesting a bifunctional action of these G proteins.
G
o-subunits are well known to modulate the activity of
Ca2+ channels (37); thus a G
o-regulated
channel may contribute to govern the membrane potential in this subset
of neurons. In the context of a bifunctional role for
G
i-subunits, it is interesting to note that vomeronasal
neurons express adenylyl cyclase subtype II (13).
G
i-subunits inhibit AC II (38), thus, it is conceivable that activation of Gi by volatile pheromonal components may
not only lead to phospholipase C activation mediated by

-subunits, but in addition to G
i controlled
inhibition of AC II. Interestingly, an attenuation of cAMP levels in
VNO preparations upon stimulation with the volatile pheromonal
components dehydro-exo-brevicomin and 2-(sec-butyl)-4.5-dihydrothiazole
has recently been reported (39).
The stereotypical coexpression pattern of the two families of candidate
pheromone receptors (14-17) with either Gi or
Go implies a functional linkage between both signal
transduction elements. Based on characteristic structural features of
each receptor family, it has been suggested that the two receptor
classes may recognize structurally different chemical stimuli (40). One
could imagine that V1Rs, characterized by short N-terminal domain and
interconnecting loops, like ORs (14), detect low molecular weight
volatiles, whereas V2Rs, having an extremely large highly variable
N-terminal domain, may recognize high molecular weight nonvolatile
pheromones. Alternatively, as V2Rs-related G protein-coupled receptors,
like metabotrophic glutamate receptors (41) and the extracellular calcium sensing receptors (42) recognize very small ligands by means of
the large N-terminal domain (43), it appears conceivable that V2Rs may
bind small pheromonal components via a "venus flytrap" mechanism as
suggested for the metabotrophic glutamate receptors, whereas V1Rs,
resembling ORs in the olfactory epithelium, may recognize volatile
odorants presented by members of the lipocalin family, e.g.
odorant-binding proteins (44). The result of the present study
demonstrating that Gi coexpressed with V1Rs is activated by
volatile ligands, whereas Go, coexpressed with V2Rs is
activated upon stimulation with the protein pheromone
2u-globulin, provide the first experimental evidence
that each of the two VNO-receptor families is indeed activated by a
distinct class of ligands. The emerging concept that V1Rs are activated
by small volatile compounds, whereas V2Rs are specialized for
interacting with protein pheromones can now be tested in experimental
studies analyzing heterologously expressed receptors.
 |
ACKNOWLEDGEMENTS |
We thank Kerstin Bach for excellent technical
assistance and Jörg Strotmann for helpful discussions and
critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft, the Human Frontier Science Program, European
Community project ERBBIO 4 CT 960593, and the Fonds der Chemischen
Industrie.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.
¶
To whom correspondence should be addressed: University
Stuttgart-Hohenheim, Institute of Physiology, 70593 Stuttgart, Germany. Fax: 0711-459-3726; Tel.: 0711-459-2274; E-mail:
boekhoff{at}uni-hohenheim.de.
 |
ABBREVIATIONS |
The abbreviations used are:
OE, olfactory
epithelium;
VNO, vomeronasal organ;
IP3, inositol
1,3,4-trisphosphate;
PAGE, polyacrylamide gel electrophoresis;
Mops, 4-morpholinepropanesulfonic acid;
GTP
S, guanosine
5'-O-(thiotriphosphate).
 |
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