Chem. Senses 25: 413-421,
2000
© Oxford University Press 2000
G Protein ß
Complexes in Circumvallate Taste Cells Involved in Bitter Transduction
University Stuttgart-Hohenheim, Institute of Physiology, D-70593 Stuttgart, Germany and 1 Unilever Research Vlaardingen, P.O.B. 114, 3130 AC Vlaardingen, The Netherlands
Correspondence to be sent to: Joachim Freitag, University Stuttgart-Hohenheim, Institute of Physiology, Garbenstrasse 30, D-70593 Stuttgart, Germany. e-mail: freitag{at}uni-hohenheim.de
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Abstract |
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G protein ß
(Gß) complexes are considered to play an important role in second messenger signaling of phospholipase C (PLC). Monitoring the inositol 1,4,5-trisphosphate (IP3) response in circumvallate tissue homogenates upon stimulation with denatonium benzoate, it was demonstrated that a glutathione S-transferase–GRK3ct fusion protein—a Gß scavenger—attenuates the bitter tastant-induced second messenger reaction. Towards an identification of the Gß complex involved in rat bitter taste transduction, it was found that the G protein ß3 subtype is specifically expressed in taste receptor cells of circumvallate papillae. Gß3-specific antibodies blocked the denatonium benzoate-induced IP3 formation in a dose-dependent manner; the inhibitory effect was reversed by preincubation with the antigenic peptide. A less pronounced inhibition was observed using Gß1-specific antibodies. Analyzing individual taste cells by single cell reverse transcriptase–polymerase chain reaction approaches, overlapping expression patterns for PLCß2, G
gust, Gß3 and G3 could be demonstrated. Furthermore, the co-expression of all profiled signal transduction components in individual taste receptor cells could be detected. These data support the concept that the denatonium benzoate-induced IP3 response is mediated by an activation of PLCß2 via a Gß complex, possibly composed of Gß3 as the predominant ß subunit and G3, and imply that multiple second messenger pathways may exist in individual taste receptor cells. |
Introduction |
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Given the chemical diversity of taste molecules, complex mechanisms are involved for their transduction, ranging from amiloride-sensitive ion channels and several types of K+-channels to G protein-coupled receptors (Lindemann, 1996a
). The perception of bitter stimuli is considered to be mediated by G protein-coupled reaction cascades involving the activation of at least two different effector enzymes (Kinnamon and Margolskee, 1996). One of the proposed mechanisms for signaling bitter tastants is the activation of phosphodiesterase (PDE), resulting in reduction of cAMP levels which cause activation of cyclic nucleotide-inhibited ion channels, thus leading to depolarization of bitter-reactive gustatory cells (Kolesnikov and Margolskee, 1995; Ruiz-Avila et al., 1995; Wong et al., 1996). In addition, there is compelling evidence for an alternative mechanism for bitter transduction which relies on activation of phospholipase C (PLC) leading to generation of IP3 (Akabas et al., 1988; Hwang et al., 1990; Spielman et al., 1994). A special subtype of PLCß has recently been identified from rat circumvallate papillae which is likely to participate in sensory transduction of the bitter agent denatonium benzoate (Rössler et al., 1998). Since this isoform is closely related to human PLCß2, it may be activated in the same manner as proposed for the human isoform (Camps et al., 1992; Park et al., 1992; Sankaran et al., 1998). In cotransfection assays, receptor-mediated release of G protein ß (Gß) subunits from pertussis toxin-sensitive heterotrimeric G proteins was shown to result in activation of human PLCß2 (Katz et al., 1992). Possible sites for interaction with Gß complexes are the pleckstrin homology domain, a region separating the bipartite catalytic domain and the first half of the Y box (Wu et al., 1993; Lee and Rhee, 1995; Kuang et al., 1996; James and Downes, 1997).
Considering a role of Gß complexes in regulating PLCß2 activity, we set out to identify and to characterize ß subunits possibly involved in rat bitter transduction.
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Isolation of taste buds, single taste cells and non-sensory lingual tissue
Tongues were isolated from freshly decapitated adult Sprague–Dawley rats (Charles River, Sulzfeld, Germany) and stored in ice-cold tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4) for 10 min. Circumvallate papillae were collected using glass capillaries and immediately frozen in liquid nitrogen (Spielman et al., 1989). Circumvallate sheets were prepared as described previously (Striem et al., 1991; Bernhardt et al., 1996; Kretz et al., 1999). Single taste buds were harvested by suction with a 40 µm pipette containing low-Ca2+ tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM EDTA, 10 mM HEPES, pH 7.4) by observation under an inverted microscope (Olympus IX70). For the isolation of single taste cells, individual taste buds were transferred to a Petri dish covered with cured Sylgard containing low-Ca2+ tyrode solution. Cells were observed under inverted microscope and taste receptor cells were identified by their characteristic morphology. Single taste cells were picked up under microscopic control with a micromanipulator by applying suction on a 10 µm microcapillary containing low-Ca2+ tyrode solution. Single cells were then transferred to PCR tubes by applying positive pressure, immediately frozen in liquid nitrogen and stored at –70°C. Non-sensory lingual tissue was obtained from epithelium around the circumvallate papilla and from the dorsal part of the tongue, just anterior to the circumvallate papilla, including underlying muscle and salivary tissue.
RNA preparation, cDNA synthesis and amplification of cDNA
Total RNA isolated from non-sensory control tissue was prepared by tissue homogenization in TRIzol reagent (Gibco BRL, Eggenstein, Germany) according to the manufacturer’s instructions, including a DNase digestion (DNase I, Gibco BRL). Subsequently, mRNA was isolated from total RNA of control tissue and directly from single taste buds (Dynabeads, Dynal, Oslo, Norway), and reverse transcribed using a first-strand cDNA synthesis kit according to the manufacturer (Pharmacia, Freiburg, Germany). cDNA of individual taste buds was synthesized and re-amplified as described in the following for single taste cells. For lysis of single cells, frozen cells were incubated at 72°C for 2 min and rapidly chilled on ice. Single-stranded cDNA was synthesized using Superscript reverse transcriptase II (Gibco BRL) according to the manual provided with the SMARTTM cDNA technology (Clontech Laboratories, Heidelberg, Germany). The amplification of the cDNA was performed using the SMARTTM PCR cDNA synthesis kit (Clontech Laboratories) according to the manufacturer’s recommendations. Cycling parameters were as follows: 95°C for 1 min; 95°C for 15 s, 65°C for 30 s and 68°C for 6 min (24 cycles); 68°C for 6 min. After ethanol precipitation of the amplified cDNA the resulting pellet was resuspended in 20 µl water.
Reverse transcriptase–polymerase chain reaction (RT–PCR), cloning and sequencing
For amplification of genes encoding Gß subtypes, equal amounts (20–100 ng) of cDNA from single taste buds or non-sensory control tissue was used as the template. For analysis of Gß gene expression, five independent amplifications were performed: four experiments employing an individual taste bud and one experiment with a pool of six taste buds. The sense primers were for Gß1 5'-TGACACCAGACTGTTTGTCTC-3'; for Gß2 5'-CCAGATCACAGATGGGCTG-3'; for Gß3 5'-AGAAGACAGTGTTCGTGGGAC-3'; for Gß4 5'-GGGGATATGATTCCAGGCTAC-3'; and for Gß5 5'-TACAGAGCTTCCATGGGCA-3'. The antisense primers were common for Gß1–4 5'-KCCWGTDGCCACAGCCATSC-3' and for Gß5 5'-GATCCCATGATCCCGAGCAG-3'. Amplification was carried out in 50 µl of 10 mM Tris–HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 25 pmol of each primer and 2 U of Taq DNA polymerase (Gibco BRL). PCR was performed according to the following conditions: 94°C for 1 min; 94°C for 30 s, 62.5°C for 1.30 min and 72°C for 40 s (25 cycles); 72°C for 7 min. A 0.5–1 µl aliquot of the re-amplified cDNA from a single cell was used as template for specific RT–PCR amplifications. In all, 12 individual taste cells were subjected to single cell RT–PCR. PCR was carried out as described before. The specific primers for PLCß2 were 5'-CTGGAGGCTGAAGTAAAGGAG-3' (sense) and 5'-GCCCCTGCATGTATGTTAGG-3' (antisense); for Gß3 5'-GGACTCTCTGAAGTGTGAG-3' (sense) and 5'-CACCTATAGTCATGGGCTG-3' (antisense); and for Ggust 5'-CAATCCGAGAAGTAGAGAGG-3' (sense) and 5'-GCTGTTGAAGAGGTGAAGAC-3' (antisense). In order to identify G subtypes present in single cells, degenerate primers matching conserved regions of G coding genes were used which had been tested for their ability to amplify common G subtypes (Gallagher and Gautam, 1994; Gautam et al., 1998). The primers were 5'-GTIGAICARCTIAARATI-3' (sense) and 5'-YTCICKRAAIGGRTTYTC-3' (antisense). The amplification was performed according to the following schedule: 94°C for 2 min, 50/60°C for 2 min and 72°C for 5 min (1 cycle); 94°C for 30 s, 50/60°C for 1.30 min and 72°C for 30 s (34 cycles); 72°C for 7 min. The annealing temperature for PLCß2, Gß3 and Ggust was 60°C; for genes encoding G subtypes it was 50°C. To exclude an amplification of genomic DNA contamination, the quality of cDNA was monitored by using primers corresponding to distinct exons of the ß actin gene in PCR reactions (Ziegler et al., 1992). Actin exon 4 primer (5'-TCATGTTTGAGACCTTCAA-3') and actin exon 5 primer (5'-GTCTTTGCGGATGTCCACG-3') amplified a 512 bp fragment transcribed from RNA and a 607 bp fragment from genomic DNA. Following PCR, 10 µl of the reaction products were separated on 1.2% agarose gels. PCR products were subcloned into pGEM-5Zf(+) vector using the pGEM-T vector system I (Promega, Mannheim, Germany). Recombinant plasmids were subjected to DNA sequencing using the RR Dye Deoxy Terminator cycle sequencing kit (PE Biosystems, Weitherstadt, Germany). Automatic sequencing was performed on an ABI 310 sequencer (PE Biosystems).
Analysis of sequence data
Analysis of sequence data was performed using HUSAR 3.0 software package based on the sequence analysis software package 7.2 from the Genetic Group (Madison, WI).
Stimulation experiments and second messenger determination
The circumvallate papilla from each rat tongue was dissected using glass capillaries and immediately frozen in liquid nitrogen (Spielman et al., 1989, 1996). For a typical stimulation experiment, 10 circumvallate papillae were minced in a glass homogenizer in hypotonic buffer (10 mM Tris/HCl, 3 mM MgCl2, 2 mM EGTA, pH 7.4) and centrifuged at 750 g for 10 min at 4°C. The supernatants were immediately employed in subsequent stimulation experiments. Therefore, denatonium benzoate was diluted in reaction buffer [200 mM NaCl, 10 mM EGTA, 50 mM 4-morpholinepropanesulphonic acid, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.05% sodium cholate, 1 mM ATP and 4 µM GTP, 12 nM free calcium calculated and adjusted as described elsewhere (Pershadsingh and McDonald, 1980), pH 7.4] containing denatonium benzoate at a final concentration of 100 µM. To prevent degradation of IP3, stimulation experiments were performed in the presence of 20 mM LiCl (final concentration during incubation). The reaction was started by mixing 210 µl of prewarmed reaction buffer with 50 µl of the protein samples (0.59 ± 0.1 µg/µl), incubated for 2 min at 37°C in a shaking water bath and stopped by 7% ice-cold perchloric acid (105 µl) before the concentration of IP3 was determined according to Palmer and Wakelam (Palmer and Wakelam, 1989). To determine the influence of the glutathione S-transferase (GST)–GRK3ct fusion protein, the GST control peptide and the subtype-specific Gß antisera (Santa Cruz Biotechnology, Santa Cruz, CA) on the stimulus-induced second messenger responses, circumvallate protein samples were preincubated with the indicated dilution of the modulators for 10 min on ice. The GST–GRK3ct fusion protein and the GST control peptide were prepared and purified in a manner described previously (Koch et al., 1993). In additional experiments, the specificity of the Gß3-specific antibodies was verified by incubating these antibodies with the corresponding peptide according to the specifications of the manufacturer (Santa Cruz Biotechnology) prior to stimulation.
In situ hybridization
Circumvallate papillae were dissected from 2- to 5-week-old rats, embedded in Tissue Tek (Miles Inc., Elkhart, IL) and rapidly frozen in a liquid N2 cooled isopentane bath. Coronal sections of 10 µm were cut on a Leica cryostat (model CM 3000) at –30°C and thaw-mounted on silanated slides. Generation of antisense and sense digoxigenin-labeled probes from Gß1, Gß2 and Gß3 cDNAs previously amplified by RT–PCR and subsequent in situ hybridization were performed as described previously (Rössler et al., 1998).
SDS–PAGE and Western blot analysis
Membrane fractions of the gustatory tissue were prepared for SDS–PAGE as described previously (Rössler et al., 1998), subjected to 12.5% acrylamide gel electrophoresis and analyzed using the Laemmli buffer system (Laemmli, 1970). The separated proteins were transferred onto nitrocellulose using the semi-dry blotting system (Pharmacia). The blot was stained with Ponceau S and stored at 4°C until use. For Western blot analysis, non-specific binding sites were blocked with 8% non-fat 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 the Gß3 polyclonal antibody (Santa Cruz Biotechnology) diluted in TBST, containing 3% non-fat milk powder. After three washes with TBST, horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:10 000 dilution in TBST with 3% milk powder) was applied and the enhanced chemoluminescence system (Amersham) was used to monitor immunoreactivity.
Immunohistochemistry
Circumvallate papillae were dissected from tongues of 2- to 5-week-old rats and placed in 4% paraformaldehyde, 1% picric acid in phosphate-buffered saline (PBS; 150 mM NaCl, 1.4 mM KH2PO4, 8 mM Na2HPO4, pH 7.4), pH 7.3, for 2 h at 4°C. Tissue was rinsed in PBS, cryoprotected by immersion in 25% sucrose/PBS for 2 h at 4°C and embedded as described before. Coronal sections (10 µm) adhered to Superfrost plus (Fisher, Orangeburg, NY) microslides were air-dried for 4 h. Slides were treated with 0,1% Triton X-100 in PBS for 3 min. After washing three times in PBS, unspecific binding sites were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min in a wet chamber at room temperature. Excess of blocking solution was removed before application of the Gß1-, Gß2- and Gß3-specific antibody (Santa Cruz Biotechnology), diluted 1:200 in 1% BSA/PBS. After incubation at 37°C in a wet chamber for 2 h, sections were washed three times with PBS. The goat anti-rabbit IgG conjugated to the flourescent dye Cy 3 antibody (Jackson ImmunoResearch, West Grove, PA) was applied (1:500 in PBS) and incubated for 1 h at room temperature. After three washes with PBS the reaction was stopped with double distilled water. The slides were air-dried, embedded in Vectashield (Vector Laboratories, Burlingame, CA) and examined under a Zeiss Axiphot microscope. The specificity of the immunoreactivity obtained by the Gß3-specific antibody was confirmed in control experiments employing primary antibodies pre-incubated with the antigenic peptide according to the specifications of the manufacturer (Santa Cruz Biotechnology).
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Results |
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In order to approach the question whether Gß
subunits may play a role in tastant-induced IP3 responses, a fragment of the G protein receptor kinase GRK3 was used as a scavenger for ß subunits with respect to its capacity to directly interact with ß dimers and to block their ability to modulate corresponding effectors (Pitcher et al., 1992; Boekhoff et al., 1994; Daaka et al., 1997). Therefore, tissue homogenates of circumvallate papillae were incubated with a GST–GRK3ct fusion protein containing the C-terminal 222 amino acid residues corresponding to the ß binding domain of the GRK3 (Pitcher et al., 1992) and subsequently analyzed for formation of IP3 upon stimulation with denatonium benzoate. In such second messenger assays, the bitter agent causes a generation of IP3 in taste tissue, but not in non-gustatory control tissue (Spielman et al., 1996; Rössler et al., 1998). As demonstrated in Figure 1, in the presence of GST–GRK3ct fusion protein the denatonium benzoate-induced IP3 response was significantly attenuated. In contrast, a GST control peptide had no effect on the tastant-induced second messenger response, emphasizing the specificity of the inhibitory effect. Since the C-terminal domain of GRK3 has no enzymatic activity, it is conceivable that the inhibitory effect is due to its interaction with membrane-anchored ß subunits, thus supporting the notion that Gß subunits may mediate a tastant-induced activation of phospholipase C.
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As a first step towards an identification of the distinct ß and
subunits possibly involved in the transduction process for bitter compounds, RT–PCR assays were performed to identify the ß subtypes expressed in gustatory tissue. To date, five ß subunits have been identified in mammals, generally designated as Gß1–Gß5; in addition, a heart-specific rat Gß3 subtype as well as a retina-specific Gß5 splice variant have been described (Ray and Robishaw, 1994; Watson et al., 1994; Gautam et al., 1998). Specific oligonucleotide primer pairs targeting each known ß subunit were employed in RT–PCR experiments using cDNA obtained from isolated taste buds of rat circumvallate papillae as template. For comparison, experiments with cDNA from non-sensory lingual tissue were performed. The results of these RT–PCR studies, shown in Figure 2, indicate highly amplified PCR fragments of the expected size for Gß3. In contrast, specific primers for Gß1 and Gß2 resulted in minor signals of predicted size, whereas no amplification products were detectable using either Gß4- or Gß5-specific primers. For the non-sensory lingual tissue, strong amplification signals for Gß1 and Gß2 and weak signals for Gß3 and Gß4 were obtained (Figure 2). Sequence analysis of the amplified PCR fragments confirmed their identity to the previously described Gß1, Gß2 and Gß4 subtypes, as well as to the Gß3 subtype that is highly expressed in rat heart (Ray and Robishaw, 1994).
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To explore the topological expression pattern of specific Gß subtypes in the lingual tissue, in situ hybridization experiments were performed using digoxigenin-labeled antisense probes of Gß1, Gß2 and Gß3. Only the Gß3 subtype was expressed in gustatory cells of the circumvallate taste buds (Figure 3a,b); transcripts for Gß2 were weakly detected in cells close to the basal membrane of the rat taste buds and in cells of the surrounding non-gustatory lingual epithelium (Figure 3d). The antisense probe for Gß1 failed to produce any hybridization signal within taste buds or adjacent tissue (Figure 3e). Since the RT–PCR led to the isolation of Gß1, the failure of the Gß1 antisense probe to hybridize within circumvallate buds may be due to a very low expression level.
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To localize Gß3 protein and to monitor the subcellular localization, immunohistochemical experiments were performed. The specificity of the employed antibodies was first analyzed in Western blots. As depicted in Figure 4, antibodies raised against Gß3 visualized a polypeptide band with an apparent molecular mass of 37 kDa in circumvallate membrane preparations, corresponding to the expected molecular weight for Gß3. The specificity of the labeling was confirmed by preincubation of the primary antibody with the antigenic peptide, which inhibited immunoreactivity (Figure 4). Subsequently, coronal sections through the tongue were subjected to immunostaining. It was found that the Gß3 protein was located in a subset of spindle-shaped cells; particular strong immunoreactivity was observed at the apical part of taste cells. No labeling was detectable in surrounding epithelial cells (Figure 5a). The possibility that the observed immunoreactivity represented artifactual labeling was excluded in a control experiment: when the primary antibody was blocked with the antigenic peptide, no labeling could be detected (Figure 5b). Employing specific antibodies for Gß1 and Gß2 gave no staining of elongated taste cells; Gß2 was restricted to cells in the basal region of the taste buds and low immunoreactivity for Gß1 was found in the microvillar taste pore region (data not shown). These results confirm the in situ data and suggest that Gß3 may play a role in taste signal transduction.
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To assess this notion, biochemical assays were performed monitoring the effect of Gß3-specific antibodies on denatonium benzoate-induced IP3 responses in circumvallate preparations. As shown in Figure 6, preincubation with Gß3 antibodies attenuated the bitter tastant-induced IP3 response in a dose-dependent manner. Gß3 antibodies caused a strong inhibition already at very low antibody concentrations; 47% inhibition was registered at a 1:5000 dilution. The specificity of this effect was confirmed in control experiments, employing Gß3 antibodies neutralized with the antigenic peptide, where no inhibitory effect was observed (Figure 7). Comparing the effects of the Gß subtype-specific antibodies on the denatonium benzoate-induced IP3 signaling, it was found that Gß2 antibodies did not affect the responsiveness; in contrast, antibodies specific to Gß3 and Gß1 reduced IP3 formation elicited by the bitter agent; however, at a 1:1000 dilution of both subtype-specific antibodies, the inhibitory effect of Gß1 was less pronounced than blockage observed upon pretreatment with Gß3-specific antibodies (Figure 7).
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Even though these results strengthen the hypothesis for a functional involvement of Gß3 in denatonium benzoate-induced IP3 transduction, it still remains unclear whether Gß3 could communicate bitter signals from receptors to the effector enzyme PLCß2. Therefore, individual taste cells were assessed by single-cell RT–PCR approaches in order to examine PLCß2-positive cells for expression of G protein subtypes. Twelve individual taste receptor cells were picked according to their typical elongated bipolar morphology. To monitor possible RNA degradation, cells were first tested for ß actin expression. All 12 cells showed a positive actin amplification, indicating the integrity of the employed RNA (data not shown). Of these 12 cells, eight were PLCß2 positive. These eight cells were further analyzed employing a specific primer combination for Gß3, degenerate oligonucleotides for G
subunits and primers for the taste cell-specific G protein subunit gustducin (Ggust) (McLaughlin et al., 1992; Gallagher and Gautam, 1994; Clapham and Neer, 1997; Gautam et al., 1998). In detail, two of the PLCß2-expressing taste cells showed amplification products of the expected size employing Gß3- (476 bp), Ggust- (469) and G-specific primers (140 bp) (Figure 8). Two other cells showed positive amplification with Gß3- and Ggust-specific primers, and an additional two cells with primers targeting only G subunits. Two cells showed amplification products either with Gß3- or with Ggust-specific primers. By subcloning and sequence analysis, the G-specific amplification products were identified as G3-coding sequences in each cell, whereas the specificity of the other amplification products could be confirmed.
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These results indicate that PLCß2, G
gust, Gß3 and G3 are coexpressed in individual cells and suggest that the denatonium benzoate-induced transduction cascade could be mediated by PLCß2 and the Gß33 complex. |
Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Several bitter-tasting compounds have been correlated with PLC activation and a raise of IP3 levels in gustatory cells (Spielman et al., 1996
). With respect to the sensitivity of PLC to stimulation by ß dimers (Park et al., 1993; Wu et al., 1993), the results of the present study point to a role for distinct Gß complexes as modulators of the PLC effector system. The specific expression of the Gß3 subtype in gustatory cells of circumvallate taste buds and the inhibitory effect of Gß3-specific antibodies on the denatonium benzoate-induced IP3 formation suggest a major function for this ß subunit in mediating second messenger signaling in bitter taste. So far, the rat Gß3 subtype has been found to be predominately expressed in heart, whereas the closely related human Gß3 (96% identity) is most abundantly expressed in retina and brain (Levine et al., 1990; Ray and Robishaw, 1994), suggesting that each subtype is restricted to specialized systems and interacts with a limited set of effectors.
One proposed mechanism for transduction of the intensely bitter compound denatonium benzoate is an increase of IP3 levels via activation of PLC (Akabas et al., 1988; Hwang et al., 1990; Spielman et al., 1994). The possible effector enzyme likely involved in transducing Gß-mediated responses to bitter tastants is the recently identified novel PLCß2 subtype, which is selectively expressed in distinct taste sensory cells of the rat circumvallate papillae (Rössler et al., 1998). In addition, it has been proposed that denatonium benzoate may activate an alternative transduction pathway mediated by the taste-specific G subunits gustducin and/or transducin, activating a phosphodiesterase; the resulting decreased levels of cyclic nucleotides might elicit taste cell depolarization through a cyclic nucleotide-suppressible conductance (Kolesnikov and Margolskee, 1995; Ruiz-Avila et al., 1995; Wong et al., 1996). Besides the given multiplicity of pathways involved in bitter taste transduction, the molecular data of this study demonstrate that an individual cell expressing PLCß2 is also equipped with the Gß33 complex and Ggust. The notion that a similar portion of the monitored cells express either PLCß2, Ggust and Gß3 without G3 or, alternatively, PLCß2 and G3 without Ggust and Gß3 raises the possibility that other Gß or G subunits might be involved in bitter signal transduction processes. In this context, it is interesting to note that recent studies led to the identification of a novel G subtype (G13) which is coexpressed with gustducin and which is functionally involved in bitter taste transduction (Huang et al., 1999); furthermore, it was found that gustducin-positive cells always express G13 and Gß3, whereas 79% also express Gß1. Thus, the results of the present study demonstrating that Gß3- as well as Gß1-specific antibodies block the denatonium benzoate-induced rise in IP3 led to the suggestion that distinct compositions of Gß and G subtypes might take part in bitter taste transduction processes. As distinct ß subunits have selective effects on the activity of effector enzymes (Clapham and Neer, 1997; Hamm, 1998), it is conceivable that the Gß1 and Gß3 subtypes could differ in their regulatory activities of downstream signaling molecules.
The finding that Ggust and PLCß2 are coexpressed in the same cell raises the possibility that particular taste sensory cells may respond to denatonium benzoate through the PDE- as well as the PLC-mediated signaling machinery, thus activating parallel pathways. This notion is supported by a recent study demonstrating that denatonium induces not only a rise in the IP3 level but also a suppression of the cAMP level; preincubation with gustducin antibodies rescued the suppressive effect on cAMP but did not affect IP3 responses (Yan et al., 1999). Also, in mouse fungiform papilla denatonium elicited both IP3 and cAMP responses (Nakashima and Ninomiya, 1999). These effects may account for a synchronous activation of the PDE cascade through Ggust and the PLC cascade through the associated ß complex in a distinct taste cell subpopulation. Basically, the existence of multiple second messenger pathways in one taste cell may have an important role in fine-tuning and modifying the response to a given taste stimulus, as has been suggested for the olfactory system (Ache and Zhainazarov, 1995). Moreover, it has been proposed that an individual taste cell may respond to more than one stimulus (Lindemann, 1996b). Recent evidence that particular taste cells express both putative taste receptors, TR1 and TR2, as well as the observation that neither TR1 nor TR2 are uniformly coexpressed with gustducin strengthen this concept (Hoon et al., 1999). Future studies should gain further insight how the sensory cells receive and process tastant stimuli.
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Acknowledgments |
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We thank Prof. B. Lindemann for teaching us how to isolate taste buds, K. Bach for excellent technical assistance and S. Conzelmann for help with in situ hybridization and micrographs. We are very grateful to Prof. R. Lefkowitz for generously providing the GRK3ct fusion protein and the GST control peptide. This work was supported by the Deutsche Forschungsgemeinschaft (grant FR 1444/1-2) and by Unilever, Vlaardingen, the Netherlands. P.R. is supported by the ‘Graduiertenförderung’ of the Land Baden-Württemberg; I.B. is a recipient of the ‘Margarethe von Wrangell-Habilitationsstipendium’ from the Land Baden-Württemberg.
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Accepted March 14, 2000
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