Odorants Selectively Activate Distinct G Protein Subtypes in Olfactory Cilia*

Markus SchandarDagger , Karl-Ludwig Laugwitz§, Ingrid BoekhoffDagger , Christine KronerDagger , Thomas Gudermann§, Günter Schultz§, and Heinz BreerDagger

From the Dagger  Universität Stuttgart-Hohenheim, Institut für Physiologie, 70593 Stuttgart, Germany and § Freie Universität Berlin, Institut für Pharmakologie, Thielallee 69-73, 14195 Berlin, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  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 Galpha s antibodies, whereas Galpha o antibodies blocked odor-induced inositol 1,4,5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha -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 Galpha s-like protein, whereas odorants that elicited an IP3 response led to the labeling of a Galpha o-like protein. Since odorant-induced IP3 formation was also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by beta gamma 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Galpha 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 alpha  subunits are photolabeled using the hydrolysis-resistant GTP-analogue [alpha -32P]GTP azidoanilide. Subsequent immunoprecipitation of Galpha 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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-alpha -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. [alpha -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 alpha  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-- [alpha -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 [alpha -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 [alpha -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 alpha  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 [alpha -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 alpha  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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  subunits (see Table I).

                              
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Table I
Peptide sequences used for generation of antibodies

Western blot analyses using specific antisera were performed to determine the relative distribution of distinct Galpha 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 Galpha i-1, Galpha i-2, and Galpha 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 Galpha q-specific antiserum. Western blot analysis with a Galpha o-specific antibody directed against an epitope corresponding to the sequence common to both Galpha o-1 and Galpha 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 alpha  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 Galpha s (1:1000), C-10 for Galpha i (1:2000), K-20 for Galpha o (1:3000), and AS 368 for Galpha q (1:2000). In addition, membrane fractions of cerebral cortex (Co) were assayed for G protein alpha  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.

Antibodies specific for distinct G proteins have been used successfully in functional studies. For example specific inhibition of (a) ligand-induced, alpha  subunit GTPase activity (29, 30) and (b) alpha -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-Galpha 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 Galpha s antibodies. Isolated olfactory cilia were incubated for 10 min on ice with different concentrations of a Galpha s-specific antiserum (AS 348) raised against a peptide common to both Galpha s and Galpha 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 Galpha 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 Galpha 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).

Activation of phospholipase Cbeta 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 Galpha i, Galpha q, and Galpha 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 Galpha i serum (Fig. 3B). In contrast, antibodies against Galpha 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. Galpha q antibodies gave a significant inhibition only at the highest concentration (1:100).


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Fig. 3.   Effects of different Galpha antibodies on odor-induced second messenger formation. Aliquots of isolated olfactory cilia were preincubated with different concentrations of affinity-purified subtype-specific Galpha 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.

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 [alpha -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 Galpha s proteins. Isolated olfactory cilia were incubated with [alpha -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). Galpha s subunits were immunoprecipitated using an antiserum directed against Galpha s subtypes, separated on SDS-PAGE, and the incorporated [alpha -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 Galpha 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 [alpha -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 Galpha 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 Galpha s. Membranes of isolated olfactory cilia were photolabeled with [alpha -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 Galpha s common antiserum AS 348 and subsequently subjected to SDS-PAGE. Note that, in the presence of low GDP concentrations, photolabeling of Galpha 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 Galpha s observed.

In view of the rapid kinetics of olfactory reaction cascades, time course experiments on agonist-induced labeling of Galpha 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 Galpha s-like protein was highest at short incubation times; the relative labeling was fully saturated after 10 s. In contrast, hormone-induced incorporation of [alpha -32P]GTP azidoanilide into Galpha 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 Galpha s photoaffinity labeling in isolated olfactory cilia and human platelet membranes. Membranes were incubated with [alpha -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 Galpha s constantly increased during the whole time course, reflecting the accumulation of G protein alpha  subunits liganded with [alpha -32P]GTP azidoanilide which is poorly hydrolyzed by alpha  subunits (data not shown). After solubilization, membrane proteins were immunoprecipitated with an Galpha s common antiserum (AS 348). The data represented as ligand-induced stimulation of photolabeling of Galpha s calculated as percent of maximal incorporation are a representative of three independent experiments with identical results. Data for Galpha s labeling of human platelet membranes under cicaprost stimulation were taken from Laugwitz et al. (19).

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 [alpha 32P]GTP azidoanilide into Galpha 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 Galpha 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 Galpha 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 Galpha s was immunoprecipitated with the Galpha 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 [alpha -32P]GTP azidoanilide. The autoradiogram of photolabeled Galpha 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 Galpha s by citralva as a percentage of maximal incorporation.

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 Galpha 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 [alpha -32P]GTP azidoanilide compared with samples incubated without odorant (Fig. 7A). Thus this procedure also allows for the determination of odorant-dependent Galpha s labeling by less potent adenylyl cyclase activators.


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Fig. 7.   Photolabeling of Galpha s upon stimulation with distinct odorants. A, in the presence of [alpha -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 Galpha s common antiserum AS 348, and precipitated protein was subjected to SDS-PAGE. Whereas eugenol stimulation induced enhanced incorporation of [alpha -32P]GTP azidoanilide incorporation, lyral did not affect the basal photolabeling of Galpha s. B and C, quantification of the [alpha -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 [alpha -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 Galpha s calculated as percentage of the basal photolabeling obtained in the absence of odorants. Data are the means of three independent experiments ± S.D.

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 Galpha subunits, we were able to detect odor-induced activation of Galpha s at very low odorant concentrations (see Fig. 6). Therefore, we examined the labeling of Galpha 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 Galpha 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 Galpha 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 Galpha q/Galpha 11/Galpha 14 types (AS 348) (40), Galpha i (AS 266), and Galpha 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 Galpha q or with Galpha i antibodies did not show any increase in [alpha -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 Galpha o the results were different. Whereas at low concentrations of GDP (0-100 µM) no differences were detected in photoaffinity labeling of Galpha o when compared with control samples (data not shown), at high exogenous GDP concentrations (500 µM; see Fig. 8), an odorant-induced increase in [alpha -32P]GTP azidoanilide incorporation was detected in proteins precipitated with Galpha o antibodies. This indicates that the "IP3 odors" activating Galpha o-like protein may have a similarly high nucleotide exchange rate as the Galpha 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 alpha  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 alpha  subunits of Gq/G11/G14 (AS 368) as well as antibodies directed against all alpha  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 [alpha -32P]GTP azidoanilide incorporation occurred in Galpha o.

The potency of odorants in activating the Go-like protein is demonstrated in Fig. 9A; as shown for Galpha s activation (see Fig. 6). Very low odor concentrations were sufficient to induce significant labeling of Galpha o. A densitometric evaluation of the photoaffinity labeled Galpha 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 Galpha 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 Galpha 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 [alpha -32P]GTP azidoanilide into Galpha o shown in the autoradiogram in A. Membranes were photolabeled under the conditions described in Fig. 6, and Galpha o was immunoprecipitated with the Galpha 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 Galpha o by odorants as percentage of maximal incorporation.

To evaluate the specificity of Galpha 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 Galpha o. Analyzing the intensity of the labeling densitometrically, it was clear that isovaleric acid induced a concentration-dependent increase in [alpha -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 beta gamma subunits (33), experiments were performed to explore whether the beta gamma 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 Gbeta 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 Gbeta -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 Galpha 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 Galpha o was immunoprecipitated with the Go-specific antiserum K-20. B and C, quantitative analysis of Galpha 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; Galpha o was immunoprecipitated with K-20 antiserum. Whereas isovaleric acid induced Galpha o labeling in a concentration-dependent manner, citralva and hedione did not lead to an enhanced incorporation of [alpha -32P]GTP azidoanilide. Data are presented as percentage of Galpha 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.   Gbeta antibodies attenuate odor-induced IP3 formation. A, cilia preparations were incubated with different concentrations of Gbeta 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 Gbeta 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.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The present study shows that subtype-specific antibodies attenuate odor-induced second messenger responses and immunoprecipitate activation-dependent photolabeled Galpha 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 Galpha 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 beta -type are regulated either by the alpha  subunits of the Gq family or by the beta gamma subunits of trimeric G proteins. Although the G proteins that release the activating beta gamma 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 beta gamma 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 Galpha 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.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft, the Human Frontier Science Program, EC Project ERBBIO 4 CT 960593, and the Fond 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. Tel.: 0711-459-2266; Fax: 0711-459-3726; E-mail: physiol1{at}unihohenheim.de.

1 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; Mops, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

2 I. Boekhoff, unpublished results.

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