Chemoelectrical signal transduction in
olfactory neurons appears to involve intracellular reaction cascades
mediated by heterotrimeric GTP-binding proteins. In this study attempts
were made to identify the G protein subtype(s) in olfactory cilia that
are activated by the primary (odorant) signal. Antibodies directed
against the
subunits of distinct G protein subtypes interfered
specifically with second messenger reponses elicited by defined subsets
of odorants; odor-induced cAMP-formation was attenuated by
G
s antibodies, whereas G
o
antibodies blocked odor-induced inositol 1,4,5-trisphosphate (IP3) formation. Activation-dependent
photolabeling of G
subunits with [
-32P]GTP
azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example odorants that elicited a cAMP response resulted in labeling of a
G
s-like protein, whereas odorants that elicited an
IP3 response led to the labeling of a
G
o-like protein. Since odorant-induced IP3
formation was also blocked by G
antibodies, activation
of olfactory phospholipase C might be mediated by 
subunits of a
Go-like G protein. These results indicate that different
subsets of odorants selectively trigger distinct reaction cascades and
provide evidence for dual transduction pathways in olfactory
signaling.
 |
INTRODUCTION |
Chemoelectrical signal transduction is considered to be mediated
via intracellular reaction cascades triggered by G protein-coupled receptors (1). Biochemical studies over the last decade have revealed
that odorants elicit the formation of either cAMP or IP31 in olfactory
preparations (2-6). Whereas the functional implications of the dual
transduction pathways in the crustacean olfactory system are well
established (7, 8), in vertebrates the relative importance of the two
pathways in olfactory signaling remains controversial (9, 10).
Heterotrimeric GTP-binding proteins play a key role in signal
transduction processes, coupling activated receptors to the appropriate
effector system. A variety of different G
subtypes have been
identified in vertebrate olfactory epithelium including
Gs short, Gil, Gi2,
Gi3, Go, and Gq (11-17). Even an
olfactory-specific isoform of Gs (Golf) has
been discovered (18). However, it is currently unclear how many and
which type of G proteins are involved in olfactory signal transduction.
To approach the question of which G protein subtype(s) may mediate the
transduction processes in olfactory sensory cells, it is necessary to
identify the G protein that is activated upon stimulation with distinct
odor ligands. This can be accomplished by an
activation-dependent labeling procedure (19), in which
receptor-activated G protein
subunits are photolabeled using the
hydrolysis-resistant GTP-analogue [
-32P]GTP
azidoanilide. Subsequent immunoprecipitation of G
subunits with
subtype-specific antibodies permits identification of G protein subtypes that are labeled upon stimulation of olfactory cilia preparations with distinct odorants. The data indicate that cAMP- and
IP3-inducing odorants result in labeling of different G
protein subtypes.
 |
EXPERIMENTAL PROCEDURES |
Materials
Sprague-Dawley rats were purchased from Charles River, Sulzfeld.
The odorants citralva (3,7-dimethyl-2,6-octadiennitrile), hedione
(3-oxo-2-pentyl cyclopentaneacetic acid methyl ester), eugenol
(2-methoxy-4-(2-propenyl)phenol), lilial
(para-butyl-
-methyl hydrocinnamic aldehyde), lyral
(4-(4-hydroxy-4-methyl pentyl)-3-cyclohexene-10-carboxyldehyde), and ethylvanillin (3-ethoxy-4-hydroxybenzaldehyde) were provided by
DROM, Baierbrunn. Isovaleric acid (3-methylbutanoic acid) and pyrrolidine (tetrahydropyrrole) were purchased from Sigma. The radioligand assay kits for cAMP and
myo-[3H]inositol 1,4,5-trisphosphate
determination as well as the enhanced chemoluminescence system (ECL)
were provided by Amersham Corp. [
-32P]GTP was
purchased from NEN Life Science Products. All other chemicals were
obtained from Sigma.
Methods
Antisera--
Antisera against G protein subunits were obtained
either after injection into rabbits of synthetic peptides representing
subtype-specific regions of different subunits using procedures
described previously (19, 20), or from Santa Cruz Biotechnology (Santa
Cruz, CA). In both cases, the peptide sequences used to raise the
antisera are shown in Table I.
Isolation of Olfactory Cilia--
Olfactory cilia preparations
were obtained using the calcium-shock method (21, 22). Briefly, after a
short wash of the olfactory epithelium in ice-cold saline solution (120 mM NaCl, 5 mM KCl, 1.6 mM
K2HPO, 25 mM NaHCO3, 7.5 mM glucose, pH 7.4), the tissue was subjected to Ringer's
solution containing 10 mM calcium and gently stirred for 5 min at 4 °C. Detached cilia were isolated by three sequential
centrifugation steps for 5 min at 7,700 × g. The
supernatants were collected, and the resulting pellets were resuspended
in Ringer's solution containing 10 mM CaCl2 as
described above. The cilia preparation was obtained after a final
centrifugation step of all the pooled supernatants for 15 min at
27,000 × g. The resulting pellet containing the cilia was resuspended in hypotonic buffer (10 mM Tris, 3 mM MgCl2, 2 mM EGTA, pH 7.4) and
stored at
70 °C. The yield of cilia was around 0.5 mg per rat.
Stimulation Experiments and Second Messenger
Determination--
To determine the influence of the subtype-specific
G protein
subunit antisera on the efficiency of odorant-induced
second messenger responses, isolated cilia were preincubated with the indicated dilutions of specific antisera and subsequently stimulated with an odorant mixture.
Stimulation experiments were performed at 37 °C for 2 min in the
presence of 1 mM isobutylmethylxanthine when cAMP formation was determined, or 10 mM LiCl when the IP3
response was measured. Briefly, 205 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 2 µM
GTP, pH 7.4) including 12 nM free calcium calculated and
adjusted as described elsewhere (23), with or without odorants was
prewarmed at 37 °C. The reaction was started by the addition of 30 µl (0.4-1 µg/µl) of olfactory cilia and stopped by addition of
7% ice-cold perchloric acid (100 µl) prior to determining the concentration of cAMP (24) or IP3 (25).
Photolabeling of Activated G
Proteins--
[
-32P]GTP azidoanilide was synthesized
and purified (26). Frozen cilia preparations in hypotonic buffer were
centrifuged (4 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, pH 7.4) with different GDP concentrations. The
protein concentration was adjusted to 2.6 µg/µl, and aliquots of 30 µl were adapted to 37 °C for 2 min with 20 µl of
[
-32P]GTP azidoanilide (1 µCi/tube). The reaction
was started by adding 10 µl of labeling buffer with or without
odorants. After indicated time points, the reaction was terminated by
cooling the samples to 4 °C. Excess [
-32P]GTP
azidoanilide was removed by centrifugation (4 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 prior
to addition of 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 NP-40, 10 µg/ml aprotinin,
pH 7.4), and 5 µl/tube of the indicated, nondiluted subtype-specific
G protein
subunit antisera.
After mixing for 1 h at 4 °C under constant rotation, 60 µl
of protein A-Sepharose (4 mg) were 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 NP-40, pH 7.4) and twice
with washing buffer B (100 mM Tris/HCl, 300 mM
NaCl, 10 mM EDTA, pH 7.4). Samples were then prepared for
SDS-PAGE (19). Incorporated [
-32P]GTP-azidoanilide was
determined densitometrically after gel exposure to a phosphoimager
(Fuji).
SDS-PAGE and Western Blot Analysis--
Membrane preparations
were prepared for SDS-PAGE as described previously (27), subjected to
12.5% acrylamide gel electrophoresis, and analyzed using the Laemmli
buffer system (28). For Western blot analysis, the separated proteins
were transferred onto nitrocellulose using a semidry blotting system
(Pharmacia Biotech Inc.). The blot was stained with Ponceau S and
stored at 4 °C until use. For Western blot analysis, nonspecific
binding sites were blocked with 5% nonfat milk powder (Naturaflor) in
TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and
0.05% Tween 20). The blots were incubated overnight with specific
antibodies against the different G protein
subunits diluted in
TBST, containing 3% nonfat milk powder. After three washes with TBST,
a horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000
dilution in TBST with 3% milk powder) was applied, and the ECL system
was used to monitor immunoreactivity.
 |
RESULTS |
The antisera used in this study were generated against synthetic
peptides derived from sequence domains that are unique for a particular
class of G protein
subunits (see Table
I).
Western blot analyses using specific antisera were performed to
determine the relative distribution of distinct G
subunits within
(a) a preparation of olfactory sensory cilia, (b)
total olfactory epithelium, and (c) a cerebral cortex
preparation (Fig. 1). The antiserum AS
348, which recognizes a decapeptide corresponding to the C terminus of
Gs as well as Golf (19), detected a single 44-45-kDa band. The immunoreactive polypeptide was found to be highly
enriched in olfactory cilia compared with the whole olfactory epithelium. The antibody C10, which recognized a C-terminal decapeptide of G
i-1, G
i-2, and G
i-3,
stained a single 40/41-kDa protein band in all three preparations
with similar intensity. Thus Gi subtypes are apparently not
enriched in the cilia. Similarly, labeled bands observed in both
olfactory fractions were also detected upon application of
G
q-specific antiserum. Western blot analysis with a
G
o-specific antibody directed against an epitope
corresponding to the sequence common to both G
o-1 and
G
o-2 revealed an immunoreactive band at 40 kDa that was
enriched in the cilia (see Fig. 1).

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Fig. 1.
Western blot analysis of different G protein
subtypes in olfactory tissue. To determine the relative
distribution of different G protein subunits, proteins from
isolated olfactory cilia (Ci) as well as membrane
preparations from total olfactory epithelium (Oe) (25 µg
protein) were separated by SDS-PAGE, transferred to nitrocellulose, and
subsequently probed with selective sequence-specific antibodies (AS 348 for G s (1:1000), C-10 for G i (1:2000),
K-20 for G o (1:3000), and AS 368 for G q
(1:2000). In addition, membrane fractions of cerebral cortex
(Co) were assayed for G protein subunit expression.
Immunoreactive polypeptides were visualized using the ECL system
employing conjugated horseradish peroxidase goat anti-rabbit IgG as the
second antibody. Note that Gs and Go are
enriched in olfactory cilia compared with preparations from whole
epithelium, whereas immunoreactivity of Gq and
Gi subtypes are not enriched in cilia preparations.
Molecular masses (kDa) of marker proteins are indicated.
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Antibodies specific for distinct G proteins have been used successfully
in functional studies. For example specific inhibition of
(a) ligand-induced,
subunit GTPase activity (29, 30) and
(b)
-phosphatidylinositol 4,5-diphosphate hydrolysis (31) have been described. Evidence for two second messenger pathways in
olfactory signaling suggests that more than one G protein subtype may
be involved in mediating olfactory transduction (8, 5, 32). Therefore
attempts were made to determine if the utility of subtype-specific
antibodies could be used as tools to identify G protein subtypes that
are active in olfactory signaling cascades. Isolated olfactory cilia
were pretreated with different concentrations of subtype-specific
antibodies and subsequently stimulated with odorant mixtures, which
elicit either cAMP (citralva, hedione, and eugenol) or IP3
formation (lilial, lyral, and ethylvanillin).
The effect of increasing concentrations of the anti-G
s
serum on odor-induced cAMP or IP3 formation is shown in
Fig. 2. Whereas odor-induced cAMP
formation was blocked in a concentrationdependent manner
reaching about 45% inhibition at a 1:100 dilution (Fig. 2A), IP3 formation elicited by appropriate
odorants was not significantly affected (Fig. 2B).

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Fig. 2.
Concentration-response curves for the
selective blockade of odorant-induced second messenger signaling by
G s antibodies. Isolated olfactory cilia were
incubated for 10 min on ice with different concentrations of a
G s-specific antiserum (AS 348) raised against a peptide
common to both G s and G olf. Subsequently
the samples were stimulated with odorants, and the second messenger
concentrations were determined. Only the odor-induced cAMP formation
was blocked in a concentration-dependent manner by
G s antibodies. The odor-induced IP3 signal
was not affected by this subtype-specific antiserum. Values are the
means of triplicate determinations ± S.D. Panel A
shows stimulation with a mixture of odorants inducing cAMP formation
(citralva, hedione, and eugenol, each 1 µM). The basal
cAMP level was 1733 ± 163 pmol/mg of protein; in the presence of
the highest concentration of the G s antibodies (1:100)
the level of cAMP under control conditions was 1615 ± 118 pmol/mg
of protein. Panel B shows stimulation with odorants
eliciting an IP3 response (lilial, lyral, and
ethylvanillin, each 1 µM). The basal level of
IP3 was 227 ± 67 pmol/mg of protein; pretreating
cilia with a 1:100 dilution of the antibody did not affect the
concentration of IP3 (216 ± 47 pmol/mg
protein).
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Activation of phospholipase C
subtypes is mediated by members of
either the pertussis toxin-insensitive Gq family or by
pertussis toxin-sensitive Gi and Go proteins
(33). Fig. 3 shows the effect of
increasing concentrations of G
i, G
q, and
G
o antibodies on odor-induced second messenger
responses. Pretreatment of cilia preparations with any of the three
subtype-specific antibodies did not alter the responsiveness to the
odorants citralva, hedione, and eugenol, known to induce a cAMP signal
(Fig. 3A). The IP3 response elicited by
appropriate odorants was not affected by anti G
i serum
(Fig. 3B). In contrast, antibodies against G
o significantly attenuated odor-induced IP3 formation in a
concentration-dependent manner; inhibition was 65% at a
1:250 dilution and more than 75% at a 1:100 dilution.
G
q antibodies gave a significant inhibition only at the
highest concentration (1:100).

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Fig. 3.
Effects of different G antibodies on
odor-induced second messenger formation. Aliquots of isolated
olfactory cilia were preincubated with different concentrations of
affinity-purified subtype-specific G antibodies (C-10 for
Gi, K-20 for Go, and AS 368 for
Gq); subsequently samples were stimulated with an odorant
mixture. A, effects of stimulation with a mixture of
citralva, hedione, and eugenol (each 1 µM) inducing cAMP
formation. The concentration of cAMP under control conditions
(1723 ± 299 pmol/mg) was not affected upon pretreating cilia with
the different G protein antibodies. At the highest antiserum
concentration (1:100), the level of cAMP was 1663 ± 189 for the
Gi antibody, 1685 ± 218 pmol/mg for the
Gq antibody, and 1710 ± 295 pmol/mg for the antibody
recognizing Go. Data represent the mean values of cAMP
formation (pmol/mg of protein) of triplicate determinations; the S.D.
was ± 10% or less. B, effects of stimulation by an
odorant mixture eliciting an IP3 response (lilial, lyral,
and ethylvanillin; each 1 µM). The basal IP3
level of 268 ± 25 pmol/mg of protein was unaffected, even by the
highest concentration of the G protein antibodies (Gi,
262 ± 14 pmol/mg; Gq, 275 ± 445 pmol/mg; and
Go, 242 ± 87 pmol/mg). Values are the means of
triplicate determinations ± S.D.
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With the aim of identifying directly G protein subtypes that are
activated upon odor stimulation, a photoaffinity labeling approach was
employed using the photoreactive hydrolysis resistant [
-32P]GTP azidoanilide (26, 34). Previous studies have
shown that monitoring receptor-stimulated binding of GTP analogues
require addition of exogenous GDP (19, 35, 36). Since G protein subtypes display different basal nucleotide exchange rates (37-39), it
was necessary to determine the appropriate GDP concentration that
allows visualization of odor-induced G protein labeling. In the first
set of experiments, conditions were optimized toward an odorant-induced
photolabeling of G
s proteins. Isolated olfactory cilia
were incubated with [
-32P]GTP azidoanilide in the
presence of different concentrations (0-1 mm) of exogenous GDP,
and incubation was continued for 20 s at 37 °C upon application
of a mixture of three odorant compounds (citralva, hedione, and
eugenol, each 1 µM). G
s subunits were immunoprecipitated using an antiserum directed against
G
s subtypes, separated on SDS-PAGE, and the incorporated
[
-32P]GTP azidoanilide label was determined by
autoradiography. The results of a representative experiment
(n = 3) are shown in Fig. 4. The immunoprecipitate gave a single
photolabeled band with an apparent molecular mass of 44-45 kDa, a size
identical to the molecular mass of the protein visualized in immunoblot
experiments (see Fig. 1). However, the Gs common antiserum
AS 348 used to immunoprecipitate G
s subunits does not
allow us to distinguish whether the Golf (44.7 kDa) or the
Gs short isoform (44.2 kDa), both of which are expressed
in the olfactory system, is labeled upon odorant stimulation. Comparing
the intensity of [
-32P]GTP labeling, it was clear
that, at low GDP concentrations, photolabeling is similar under control
conditions and in the presence of odorants. However, upon application
of rather high GDP concentrations (1 mM), significantly
enhanced labeling was detected in stimulated samples. This observation
contrasts with studies on photolabeling of G
s in
membrane preparations of human platelets, where agonist-induced labeling was detectable in the presence of 1 µM GDP
(19).

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Fig. 4.
Influence of GDP on photoaffinitylabeling of
G s. Membranes of isolated olfactory cilia were
photolabeled with [ -32P]GTP azidoanilide at various
GDP concentrations in the absence (control) and presence
(odor) of a mixture of citralva, hedione, and eugenol (each
1 µM). Labeled membranes were solubilized, and proteins
were immunoprecipitated with the G s common antiserum AS
348 and subsequently subjected to SDS-PAGE. Note that, in the presence
of low GDP concentrations, photolabeling of G s is
similar under control conditions and in the presence of odorants. Only
upon application of high GDP concentrations (1 mM), was a
significant odor-induced enhancement of photolabeling of
G s observed.
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In view of the rapid kinetics of olfactory reaction cascades, time
course experiments on agonist-induced labeling of G
s
were performed, in which cilia preparations were photolabeled upon incubation with an odorant mixture (citralva, hedione, and eugenol) for
different time intervals. Application of odorants elicited a rapid
incorporation of the labeled GTP analogue (Fig.
5). The ratio of agonist-stimulated to
basal photolabeling of the G
s-like protein was highest
at short incubation times; the relative labeling was fully saturated
after 10 s. In contrast, hormone-induced incorporation of
[
-32P]GTP azidoanilide into G
s of human
platelets has been shown to follow a very different time course (see
Fig. 5). Maximal labeling is reached after about 10 min.

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Fig. 5.
Time course of ligand-induced
G s photoaffinity labeling in isolated olfactory cilia
and human platelet membranes. Membranes were incubated with
[ -32P]GTP azidoanilide in the presence and absence of
either an odorant mixture containing 1 µM of citralva,
hedione and eugenol or the hormone cicaprost (1 µM) for
the times indicated in the abscissae. The basal level of
G s constantly increased during the whole time course,
reflecting the accumulation of G protein subunits liganded with
[ -32P]GTP azidoanilide which is poorly hydrolyzed by
subunits (data not shown). After solubilization, membrane proteins
were immunoprecipitated with an G s common antiserum (AS
348). The data represented as ligand-induced stimulation of
photolabeling of G s calculated as percent of maximal
incorporation are a representative of three independent experiments
with identical results. Data for G s labeling of human
platelet membranes under cicaprost stimulation were taken from Laugwitz
et al. (19).
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To determine the potency of individual odorants, photolabeling
experiments were performed using different concentrations of citralva.
As demonstrated by the autoradiogram in Fig.
6A, incorporation of
[
32P]GTP azidoanilide into G
s-like
protein increased in a concentration-dependent manner. In
addition, it is clear that even very low doses (picomolar) of the
odorant are sufficient to induce a significant labeling of
G
s. The intensity of the labeling, evaluated
densitometrically, was used to construct a concentration-response curve
(Fig. 6B). In conformity with the results of many similar
olfactory stimulation experiments, we did not detect saturation; an
approximately half-maximal activation at about 50 nM was
estimated. These results are in line with previous experiments
monitoring odor-induced second messenger responses (5).

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Fig. 6.
Concentration dependence of the citralva
effect on photolabeling of G s. A, isolated
cilia were stimulated in the presence of 1 mM GDP with
increasing concentrations of the fruity odorant citralva (1 pM to 10 µM); subsequently G s
was immunoprecipitated with the G s common antiserum AS
348. The autoradiogram showing the 45-kDa region of a SDS-PAGE is a
representative of three independent experiments each giving very
similar results. B, concentration-response curve of
citralva-induced incorporation of [ -32P]GTP
azidoanilide. The autoradiogram of photolabeled G s in
A was densitometrically evaluated. The inset C
shows the logarithm scale of odorant concentration of the dose-response
curve in B. Note that a half-maximal labeling was
accomplished at about 50 nM of citralva. Data indicate the
photostimulated luminescence of G s by citralva as a
percentage of maximal incorporation.
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Odorants showed different potencies when stimulation of adenylyl
cyclase was examined (2). To explore whether less potent adenylyl
cyclase activators also induce labeling of the G
s-like protein, cilia preparation were stimulated with the odorant eugenol, which shows only 47% of adenylyl cyclase activation compared with citralva. Stimulation of cilia preparations with 1.6 µM
eugenol caused a significant incorporation of
[
-32P]GTP azidoanilide compared with samples incubated
without odorant (Fig. 7A).
Thus this procedure also allows for the determination of
odorant-dependent G
s labeling by less potent
adenylyl cyclase activators.

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Fig. 7.
Photolabeling of G s upon
stimulation with distinct odorants. A, in the presence of
[ -32P]GTP azidoanilide isolated cilia were incubated
for 30 s without odorants (control) or with 1.6 µM of the herbaceous odorant eugenol or the fruity
odorant lyral; ciliary proteins were immunoprecipitated with the
G s common antiserum AS 348, and precipitated protein was
subjected to SDS-PAGE. Whereas eugenol stimulation induced enhanced
incorporation of [ -32P]GTP azidoanilide incorporation,
lyral did not affect the basal photolabeling of G s.
B and C, quantification of the
[ -32P]GTP azidoanilide incorporation induced by low or
high odor concentrations. Cilia membranes were stimulated with
individual odorants (citralva, isovaleric acid, and pyrrolidine) in the
presence of [ -32P]GTP azidoanilide either with very
low (16 nM) (B) or higher odor concentrations
(1.6 µM) (C). The data present the
photostimulated luminescence determined by densitometric analysis of
autoradiograms from photoaffinity labeled G s calculated
as percentage of the basal photolabeling obtained in the absence of
odorants. Data are the means of three independent experiments ± S.D.
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Several odorants have been shown not to induce a detectable cAMP signal
but rather the formation of IP3 (32). However, it might be
possible that adenylyl cyclase activation was not detected due to the
insufficient sensitivity of the method used. By photoaffinity labeling
of G
subunits, we were able to detect odor-induced activation of
G
s at very low odorant concentrations (see Fig. 6).
Therefore, we examined the labeling of G
s by individual
phospholipase C-stimulating odorants, i.e. lyral, isovaleric
acid, and pyrrolidine. The results depicted in Fig. 7, B and
C, show that application of citralva at different
concentrations induced a concentration-dependent increase
in G
s labeling, whereas neither low (16 nM)
(Fig. 7), nor high odor concentrations (1.6 µM) of isovaleric acid and pyrrolidine (Fig.
7C) or lyral (Fig. 7A) affected labeling of
G
s.
Different G protein types are known to link receptors to phospholipase
C (33). To evaluate which G protein subtype might be involved in
odor-induced IP3 formation, photolabeling studies were
performed with a stimulating odorant mixture (lilial, lyral, and
ethylvanillin, each 1 µM) followed by immunoprecipitation with subtype-specific antibodies for
G
q/G
11/G
14 types (AS 348) (40), G
i (AS 266), and G
o isoforms (AS 6)
(34). In all cases the different antibodies precipitated photolabeled
proteins with molecular masses identical to those found in the
immunoblotting experiment (see Fig. 1). However, proteins precipitated
with G
q or with G
i antibodies did not
show any increase in [
-32P]GTP azidoanilide
incorporation upon odorant stimulation, neither in the presence of a
low GDP concentration (not shown) nor in the presence of high GDP
levels (Fig. 8, 500 µM
GDP). Nevertheless, for G
o the results were different.
Whereas at low concentrations of GDP (0-100 µM) no
differences were detected in photoaffinity labeling of
G
o when compared with control samples (data not shown), at high exogenous GDP concentrations (500 µM; see Fig.
8), an odorant-induced increase in [
-32P]GTP
azidoanilide incorporation was detected in proteins precipitated with
G
o antibodies. This indicates that the
"IP3 odors" activating G
o-like protein
may have a similarly high nucleotide exchange rate as the
G
s-like G protein, which is labeled upon application of
"cAMP-odors" (see Fig. 4).

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Fig. 8.
Photoaffinity labeling of individual G
protein subunits upon stimulation with an odorant mixture inducing
IP3 formation. Membrane preparations of olfactory
cilia were incubated for 2 min in the presence of 500 µM
exogenous GDP either in the absence of odorants (control) or
in the presence of an odorant mixture comprised of lilial, lyral, and
ethylvanillin (each 1 µM) (odorant). To
identify activated G protein subtypes, immunoprecipitation of
solubilized photolabeled ciliary membranes was performed using
antibodies which recognized the subunits of
Gq/G11/G14 (AS 368) as well as
antibodies directed against all subtypes of the Gi (AS
266) or Go isoforms (AS 6). Although all three antibodies
precipitated photolabeled proteins with molecular masses identical with
those found in immunoblotting, the only odor-induced increase in
[ -32P]GTP azidoanilide incorporation occurred in
G o.
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The potency of odorants in activating the Go-like protein
is demonstrated in Fig. 9A; as
shown for G
s activation (see Fig. 6). Very low odor
concentrations were sufficient to induce significant labeling of
G
o. A densitometric evaluation of the photoaffinity labeled G
o-like protein is presented in Fig.
9B; the concentration-response curve revealed an
apparent half-maximal labeling of Go at odorant concentrations of about 200 nM.

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Fig. 9.
Concentration dependence of odorant-induced
G o labeling. A, membranes of olfactory cilia
were photolabeled in the presence of 500 µM GDP either
under control conditions or in the presence of increasing
concentrations (2 pM to 20 µM) of an odorant
mixture (lilial, lyral, and ethylvanillin). For precipitation of
G o, the antiserum AS 6 was used. Note that significant
labeling was detectable at very low odorant doses. The autoradiogram
showing the 45-40-kDa region of a SDS-PAGE is representative of three
independent experiments with similar results. B,
quantitative analysis of the concentration dependence of odor-induced
incorporation of [ -32P]GTP azidoanilide into
G o shown in the autoradiogram in A. Membranes
were photolabeled under the conditions described in Fig. 6, and
G o was immunoprecipitated with the G o
common antibody AS 6. Half-maximal labeling was accomplished at about
200 nM of the odorants. The inset C shows the
logarithm scale of odorant concentration of the dose response curve in
B. Data indicate the photostimulated luminescence of
G o by odorants as percentage of maximal
incorporation.
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To evaluate the specificity of G
o labeling, we analyzed
the effects of individual odorants that have been shown to activate phospholipase C. Stimulation with the "IP3-odorants"
lyral (Fig. 10A; 1.6 µM) or isovaleric acid (Fig. 10, B, 16 nM, and C, 1.6 µM) induced an
enhanced photolabeling of G
o. Analyzing the intensity of
the labeling densitometrically, it was clear that isovaleric acid
induced a concentration-dependent increase in
[
-32P]GTP azidoanilide incorporation. In contrast, the
application of high odor concentrations of the cAMP compound eugenol
did affect Go labeling (see Fig. 10A; 1.6 µM); even stimulation with high concentrations of the
very potent adenylyl cyclase activators citralva or hedione failed to
induce a significant incorporation of the GTP analogue (see Fig.
10C, 1.6 µM). As Gi and
Go subtypes usually activate phospholipase C through their

subunits (33), experiments were performed to explore whether the

subunit of the identified Go-like G protein is
mediating phospholipase C activation. Isolated olfactory cilia were
pretreated with an antiserum selective for the N-terminal sequence
common to all members of G
subunits; subsequently, odor-induced
second messenger responses were determined. Whereas odor-induced cAMP
formation was not affected (Fig.
11B), the odor-induced
IP3 response was attenuated by G
-antibodies; at a 1:250
antibody dilution, the odor-induced IP3 signal was reduced
to 30% (Fig. 11A).

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Fig. 10.
Photoaffinity labeling of G o
upon stimulation with individual odorants. A, olfactory
cilia were photolabeled for 30 s either in the absence
(control) or the presence of 1.6 µM lyral or
eugenol; subsequently G o was immunoprecipitated with the
Go-specific antiserum K-20. B and C,
quantitative analysis of G o photolabeling upon
stimulation with different concentrations of individual odorants.
Membranes of olfactory cilia were stimulated either with low (16 nM) (B) or high (1.6 µM)
(C) concentrations of isovaleric acid, citralva, or hedione;
G o was immunoprecipitated with K-20 antiserum. Whereas
isovaleric acid induced G o labeling in a
concentration-dependent manner, citralva and hedione did
not lead to an enhanced incorporation of [ -32P]GTP
azidoanilide. Data are presented as percentage of G o
labeling on photostimulated luminescence in the absence of odorants and
are the means of three independent experiments ± S.D.
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Fig. 11.
G antibodies attenuate odor-induced
IP3 formation. A, cilia preparations were
incubated with different concentrations of G antibodies
and subsequently stimulated with an odorant mixture of lilial, lyral,
and ethylvanillin (each 1 µM); basal
IP3-level of 280 ± 43 pmol/mg protein was not
affected upon pretreating cilia with the highest concentration of the
antibody (257 ± 49 pmol/mg). B, pretreating isolated
olfactory cilia with G antibodies did not affect the
odor-induced cAMP signal induced by an odorant mixture containing
citralva, hedione, and eugenol (each 1 µM); basal cAMP
level, 2313 ± 310 pmol/mg of protein; basal level of cAMP upon
pretreating cilia with a 1:100 dilution of the antibody, 2305 ± 257 pmol/mg of protein. Data are the means of three independent
experiments ± S.D.
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DISCUSSION |
The present study shows that subtype-specific antibodies attenuate
odor-induced second messenger responses and immunoprecipitate activation-dependent photolabeled G
subunits. Both
approaches indicate that a Gs-like protein mediates
odor-induced cAMP, whereas a Go-like protein controls
odor-induced formation of IP3. Thus, we have demonstrated
that different subsets of odorants selectively activate one of the two
G proteins. This finding is consistent with previous biochemical
studies indicating that odorants elicit either a cAMP or an
IP3 response in olfactory cilia preparations. Therefore,
these results provide further evidence that the phenomenon of dual
transduction pathways in olfactory signaling, which is well established
for the lobster (8), is also found in vertebrates. However, it is
presently unclear how these biochemical results can be reconciled with
the observation that transgenic mice lacking a functional cyclic
nucleotide-activated cation channel displayed general anosmia (10). The
Gs common antiserum AS 348 used to immunoprecipitate
G
s subunits did not allow us to distinguish whether
Golf (44.7 kDa) or the olfactory Gs short
isoform (44.2 kDa) is labeled upon odorant stimulation. Although it
appears likely that Golf is involved in the cAMP pathway
(41), the identity of the Gs subtype photolabeled upon odor
stimulation remains to be determined. Phospholipases of the
-type
are regulated either by the
subunits of the Gq family
or by the 
subunits of trimeric G proteins. Although the G
proteins that release the activating 
subunits are not
identified, there is evidence, that they are subtypes of the pertussis
toxin-sensitive Gi/Go family (33). The
involvement of Go proteins in phospholipase C regulation
was first observed in experiments on Xenopus oocytes
demonstrating that Go proteins specifically enhance the
Cl
current elicited by muscarinic receptors via
IP3 and Ca2+ (42-44). The observation that
olfactory phospholipase C activation occurs through 
subunits of
a Go-like G protein is thus of particular interest and in
line with previous studies indicating that odorant-induced IP3 formation in rat olfactory cilia is mediated by a
pertussis toxin-sensitive G protein (5). Although a firm identification of the Go-like protein in olfactory cilia was not possible
in this study, future investigations using more specific antibodies may
allow us determine whether one of the two previously identified Go isoforms (45, 46) or a novel G
subtype, which shares
epitopes with Go protein, is active in olfactory neurons.
In this context it is interesting to note that Go proteins
are also thought to be involved in mammalian pheromone signaling by
chemosensory neurons of the vomeronasal organ (47, 48). The observation
that two different G proteins are active in chemosensory neurons of the rat finds its parallel in the nematode Caenorhabditis
elegans. Recent findings have demonstrated that two different G
proteins encoded by gpa-2 and gpa-3 (49, 50) are
expressed in the ciliated chemosensory amphid neurons and are involved
in pheromone detection (51). Interestingly, one of the two subtypes
(GPA-3) comprises a conserved cysteine residue near the carboxyl
terminus, which is considered to be a substrate for the pertussis
toxin-catalyzed attachment of an ADP-ribose moiety to Go
and Gi proteins (52).
The rapid kinetics of ligand-induced GTP incorporation in olfactory
cilia, when compared with endocrine cells (Fig. 5), suggest a
particularly efficient interaction between the signaling molecules, notably the activated receptors and G proteins. This is reminiscent of
the high speed activation described for the rhodopsin/transducin system
(53), which may be based on fast lateral diffusion rates due to a
special lipid composition of the membrane (54). Alternatively, response
time may not be diffusion-limited, but rather elements of the
transduction machinery may be organized as architectually and spatially
distinct ultramicrodomains. Such "transducisomes" have recently
been described for the fly photoreceptors (55). Caveolae, specialized
microdomains in the plasma membrane, appear to be another compartmental
basis for a rapid and efficient coupling of transmembrane signaling
events (56, 57). Preliminary studies revealed that caveolin, a
characteristic integral membrane protein that acts as an oligomeric
docking site for distinct proteins of signaling cascades, is indeed
present in olfactory sensory cilia,2 suggesting that such
transduction centers might provide a basis for the rapid kinetics of
second messenger signaling in olfaction.