Chemical Senses

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Chem. Senses 25: 525-531, 2000
© Oxford University Press 2000

Comprehensive Study on G protein -Subunits in Taste Bud Cells, with Special Reference to the Occurrence of Gi2 as a Major G Species

Yuko Kusakabe¹, Akihito Yasuoka², Misaki Asano-Miyoshi^2,3, Kyoko Iwabuchi, Ichiro Matsumoto, Soichi Arai⁴, Yasufumi Emori³ and Keiko Abe

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, ² Bio-oriented Technology Research Advancement Institution, ³ Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and ⁴ Department of Nutritional Science, Faculty of Applied Biological Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan ¹ Present address: National Food Research Institute, 2-1-2 Kannondai, Tsukuba-shi, Ibaraki 305-8642, Japan

Correspondence to be sent to: Keiko Abe, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. e-mail aka7308{at}mail.ecc.u-tokyo.ac.jp

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Previous studies have identified many cDNA species that encode a variety of G protein -subunits occurring in taste buds. These include the cDNA encoding a taste-bud-specific G, gustducin (G_gust). Here we carried out comprehensive analyses of G species that occur in the taste buds of rat circumvallate papillae and also in their single cells isolated from the taste buds. Reverse transcriptase–polymerase chain reaction showed the presence of 10 kinds of G cDNAs, including a splice variant of Gs, among which G_gust, Gs, Gi2 and Gi3 cDNAs were shown to be major species. In situ hybridization and immunohistochemistry showed that Gi2, as well as G_gust, expressed in a subset of taste bud cells, and the frequency of Gi2-expression appears to be higher than that of G_gust. Southern analyses of the amplified cDNA from single cells showed that each taste bud cell expresses multiple G mRNA species. For example, some Gi2-positive cells also express one or more other G species, including Gs, Gi3 and G_gust, and there is no apparent correlation in expression among the three G species.

	Introduction

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In vertebrates, taste chemoreception takes place in taste bud cells (Lindemann, 1996). These comprise at least three types of elongated, spindle-shaped cells and a type of globular basal cell, all of which are derived from epithelial cells (Stone et al., 1995). These cell types are thought to differ physiologically as well as morphologically (Delay et al., 1986; Lindeman, 1996).

Sweet and bitter taste senses in humans and other mammals are believed to be mediated by a G-protein-coupled signaling system (Akabas et al., 1988; Avenet et al., 1988; Striem et al., 1989; Brand et al., 1991; Spielman et al., 1994) that involves G-protein-coupled receptors (GPCRs), coupling G proteins, related effectors and other intracellular signaling factors. Various molecules have been identified and proposed as constituents involved in the system. Many rhodopsin-type GPCRs (Abe et al., 1993a,b; Matsuoka et al., 1993; Tal et al., 1995; Thomas et al., 1996) and taste-specific Ca²⁺-sensing receptors (TR1 and TR2) (Hoon et al., 1999) have been isolated from tongue epithelia. Meanwhile, G_gust, a Gi-type G protein -subunit closely related to transducin (Gt), has been recognized as an essential component involved in sweet and bitter taste transduction (McLaughlin et al., 1992; Wong et al., 1996). Also, there are reportedly other G species in taste tissues (McLaughlin et al., 1992, 1994; Kusakabe et al., 1998), although their physiological significance has yet to be elucidated. The possibility exists that these G species may function in parallel with G_gust-mediated taste signaling. Relevant to this, it has been postulated that Gq and G15 mediate Ca²⁺ signaling (Kusakabe et al., 1998) in parallel with a G_gust-mediated cAMP pathway (McLaughlin et al., 1994). It is also possible that G species may be involved in other signaling pathways, such as a cell-to-cell communication between taste bud cells. However, the total understanding of such receptor-mediated signalings in taste bud cells requires comprehensive studies to describe the G proteins in taste bud cells to be performed.

In this study, we adopted four methods to analyze the expression of G species in taste buds and their constituent cells: reverse transcriptase–polymerase chain reaction (RT– PCR), in situ hybridization, immunohistochemistry and Southern analyses using amplified cDNA from single taste bud cells. By these procedures we have newly identified two G subunits, Gi2 and Gi3, as relatively abundant G subunits, in addition to the Gs and G_gust already reported (McLaughlin et al., 1994). We also show that Gi2-expression occurs in a subset of taste bud cells, suggesting the presence of a Gi2-mediated signaling pathway in the taste bud.

	Materials and methods

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Isolation of taste buds and single cells from taste buds

Five-week-old male rats (Wistar) were killed by injection of sodium pentobarbital. Tongues were dissected immediately and put into Ringer solution (150 mM NaCl, 4.7 mM KCl, 3.3 mM CaCl₂, 0.1 mM MgCl₂, 2 mM HEPES and 7.8 mM glucose). Ringer solution containing Type I collagenase (2 mg/ml, Sigma-Aldrich) (collagenase solution) was injected beneath a circumvallate papilla. After incubation for 30 min at room temperature, epithelia containing the circumvallate papillae were peeled off. Epithelial sheets were again soaked in collagenase solution for 10 min at room temperature, transferred to Ca²⁺-free Ringer solution containing 2 mM EDTA and incubated for 10 min. Taste buds were isolated in Ringer solution containing 0.1% bovine serum albumin (fraction V, Sigma-Aldrich) (BSA solution) by suction through a 50-µm-diameter pipette and transferred into a new plate containing BSA solution. The isolated taste buds were dispersed by pipetting and individual spindle-shaped cells were collected for single cell analyses.

Identification of G members expressed in taste buds

mRNA was purified from 100 taste buds by the usual method (Sambrook et al., 1989). RT–PCR was carried out using the following primers: 5'-GGIAA(AG)(AT)(GC)IACITT(TC)(CA)TIAA(AG)CA(AG)ATG-3' (primer A), and 5'-GGIAA(AG)AG(TC)ACIATIGTIAA(GA)CA(GA)ATG-3' (primer B) for sense, and 5'-(TC)(TC)ITGICCICCIAC(GA)TC(GA)AA(GC)AT-3' (primer C) and 5'-TCI(TG)(CA)IC(TG)(CT)TGICCICCIAC(GA)TC-3' (primer D) for antisense. These primers were made based on the amino acid sequences GKSTFLKQM, GKSTIVKQM, M/IFDVGGQ/HR/E and DVGGQRS/DE, which were found as consensus sequences common to most of the known G members. PCR was carried out for 30 cycles under the following conditions: 94°C for 1 min, 52°C for 1 min and 72°C for 2 min. The resulting 480 bp fragments were gel-isolated, cloned into pBluescrip SK (Stratagene) and sequenced.

RT–PCR of the mRNA obtained from single taste bud cells, and Southern blot analysis of G cDNAs

The cDNA from each of the single cells was synthesized and amplified by the method of Dulac and Axel (Dulac and Axel, 1995). Aliquots of cDNA samples prepared from single cells were electrophoresed in a 1% agarose gel and blotted onto nylon membranes (Hybond-N, Amersham, UK). The membranes were processed for hybridization using ³²P-labeled DNA probes corresponding to the 3'-noncoding regions of Gs, Gi2, Gi3, G_gust and ß-actin mRNAs. The 3'-noncoding regions of Gs, Gi2, Gi3 and G_gust were obtained by RT–PCR using the taste bud mRNA as a template and the specific primers, 5'-CACCTGAATTCTATGAGCAT-3' (+429–+448) and 5'-TTAAGCTTACTAAATTTGGA-3'(+1375–+1356) for Gs (Jones et al., 1987); 5'-ATAAGCTTCGGGGCAGTGGGCCTGGCAGG-3'(+1068–+1087) (added HindIII site italicized) and 5'-ATGAATTCGCCTCCAAGCGGCAGAGAGT-3' (+1517–+1498) (added EcoRI site italicized) for Gi2 (Jones et al., 1987); 5'-ATAAGCTTGAGGATGGCATAGTAAAAGC-3' (+1066–+1085) (added HindIII site italicized) and 5'-TCTCTAGAGTGGAATATTACTCCTGACC-3' (+1584–+1565) (added XbaI site italicized) for Gi3 (Jones et al., 1987); and 5'-ATGCCGTGACAGATATAATA-3'(+1027–+1047) and 5'-TGCTTGTGGCAGCCTAATTG-3' (+1510–+1491) for G_gust (McLaughlin et al. 1992). The ß-actin DNA fragment was obtained by RT–PCR using two primers, 5'-GAACCCTAAGGCCAACCGTG-3'(+1658–+1677) and 5'-ATGGTGGTGCCACCAGACAG-3' (+2775–+2794) (Nudel et al., 1983). Hybridization and washing were carried out under the usual stringent conditions at 68°C, with the final wash done in 0.1 x SSC containing 0.1% sodium dodecyl sulfate at the same temperature.

In situ hybridization

Digoxigenin-labeled anti-sense RNA probes for Gs, Gi2, Gi3 and G_gust were synthesized using T3 or T7 RNA polymerase. The circumvallate papillae layer from the tongue of a 5-week-old male rat (Wistar) was removed, frozen in OCT compound, and sectioned into 10 µm slices. In situ hybridization was performed as described previously (Schaeren-Wiemers and Gerfin-Moser, 1993; Asano-Miyoshi et al., 1998; Yasuoka et al., 1999). The sections were post-fixed for 10 min in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.0, washed twice in PBS containing 1% diethylpyrocarbonate for 15 min, washed in 5 x SSC for 15 min and prehybridized in prehybridization solution containing 50% formamide and 5 x SSC, 40 µg/ml denatured herring sperm DNA for 1 h at room temperature. Next, 0.1 ml of hybridization buffer containing 50% formamide, 5 x SSC, 5 x Denhardt’s solution, 500 µg/ml sonicated, denatured herring sperm DNA, 250 µg/ml torula tRNA, 1 mM dithiothreitol, and 5–10 ng of probe were added to the slide. The slide was covered with a siliconized coverslip and hybridized for 36 h at 58°C. The coverslips were removed by soaking in 5 x SSC at 58°C and the slides were washed twice in 0.2 x SSC for 30 min at the same temperature. The sections were washed in Tris-buffered saline (TBS) for 5 min, blocked in 0.5% blocking reagent (Boehringer) in TBS for 1 h at room temperature and incubated in the same solution containing a 1:1000 dilution of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase for 1 h. The slides were then washed three times in TBS for 15 min and once in alkaline phosphatase buffer containing 100 mM Tris–HCl, pH 9.5, 100 mM NaCl and 50 mM MgCl₂ for 5 min. The color reaction was performed by adding the color developing solution containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphatase buffer onto the slide.

Immunostaining of circumvallate papillae

Anti-Gi2 antibody, a gift from Dr T. Asano (Ueda et al., 1998), was further purified by preabsorption with recombinant G_gust protein. Anti-G_gust antibody was purchased from Santa Cruz Biotechnology Corp. We produced Gi1, Gi2 and Gi3 proteins by expression in Escherichia coli. We confirmed by Western blotting that anti-Gi2 and anti-G_gust antibodies cross-reacted only with Gi2 and G_gust, respectively. Tongues were fixed in Bouin’s solution fixative (Sigma), dehydrated, embedded in paraffin and sectioned at 6 µm thickness. Each section was immunoreacted with each of the anti-Gi2 antibody and anti-G_gust antibody, at dilutions of 1:50 and 1:100 respectively. After rinsing in PBS, the immunoreacted sections were incubated with biotin-labeled anti-rabbit IgG at a dilution of 1:500 and then treated with a complex of avidin-and-biotin-conjugated peroxidase (Vector Laboratories). The signals were developed by treatment with 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in 10 mM sodium phosphate, pH 7.4.

	Results

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Identification by RT–PCR of multiple G species expressed in taste buds

We carried out RT–PCR using the cDNAs prepared from isolated taste buds and identified 10 kinds of G cDNA clones (Table 1). Among them, G_gust, Gq and G15 have been reported to exist in taste buds (McLaughlin et al., 1992; Kusakabe et al., 1998). We newly identified an exon 3-splice variant (Gs3) that lacks part of the N-terminal region of Gs (Feinstein et al., 1992). In addition, we identified three Gi species—Gi1, Gi2 and Gi3— and a Gq species—G14—although the frequencies of Gi1 and G14 in the cDNA clones were lower. Since the cDNA used was synthesized from isolated taste buds freed from adjacent tissues, these G species identified were derived from taste bud mRNA, and the numbers of cDNA clones of each cDNA species may reflect the relative abundancy of Gs species occurring in taste bud cells.

View this table: [in this window] [in a new window]   Table 1 Number of taste bud G cDNA clones isolated by RT–PCR using two different primer sets

 
Next, in order to examine the cellular localization of each G species in taste buds, we carried out in situ hybridization experiments using the G probes obtained above. The results show that the antisense probes for Gs, Gi2, Gi3 and G_gust, all of which show higher values in Table 1, gave significant signals (Figure 1). On the other hand, the other probes gave faint signals in taste buds, and in some cases showed ubiquitous signals throughout the non-sensory epithelial tissues (data not shown). The Gs probe gave distinct signals in taste buds, but there was no specific pattern (Figure 1A). The signals for Gi3, although its expression frequency is similar to the others, were generally weak and ubiquitous in the basolateral regions of both taste buds and other parts of the epithelia (Figure 1C). Gi2 and G_gust showed characteristic expression features (Figure 1B,D). The signals for Gi2 were observed specifically in taste buds, but not in papillary epithelium lacking taste buds. The profile of G_gust was essentially the same as previously reported (Yang et al., 1999). Gi2-positive cells appeared to be larger in number than G_gust-positive cells.

View larger version (114K): [in this window] [in a new window]   Figure 1 In situ hybridization showing the expression of Gs, Gi2, Gi3 and Ggust in circumvallate papillae. Coronal sections, 10 µm each in thickness, were prepared from the circumvallate papillae of a 5-week-old male rat. Each section was hybridized with digoxigenin-labeled probes for the detection of Gs (A), Gi2 (B), Gi3 (C) and Ggust (D). Note that Gi2 (B) as well as Ggust (D) are distinctly expressed in a subset of taste bud cells. Bars represent 50 µm.

 
Since our in situ hybridization experiment showed that Gi2 and G_gust show taste-bud-specific expression (Figure 1), we next performed immunostaining with specific antibodies and compared the results with those of in situ hybridization experiments. As shown in Figure 2, subpopulations of taste bud cells were stained by both in situ hybridization and immunostaining. The results from both experiments are essentially the same. Gi2-positive cells were more than G_gust-positive cells, although the signals for G_gust were higher and clearer than those for Gi2. Gi2- and G_gust-positive cells are estimated to be 30 and 20%, respectively, in the taste bud cells.

View larger version (105K): [in this window] [in a new window]   Figure 2 In situ hybridization and immunostaining showing the expression of Gi2 and Ggust in taste buds. Conditions for in situ hybridization, methods for preparing coronal sections and details of the hybridization are the same as in Figure 1. For immunostaining, circumvallate papillae were excised from a 5-week-old male rat and fixed in Bouin’s solution. Each section, 6 µm in thickness, was immunoreacted with anti-Gi2 antibody (1:50) or anti-Ggust antibody (1:100). Signals are observed in particular cells that constitute each taste bud. The frequency of the Gi2 signals in taste buds appears to be larger than that of Ggust signals. Bars represent 10 µm.

 
Multiple G species expressed in single taste bud cells

As shown above, at least four G species (Gs, Gi2, Gi3 and G_gust) are expressed in substantial amounts in taste buds, and their expression profiles appear to differ from one another. To obtain more precise information about G expression at the cellular level, we examined how many and which G species are expressed in individual cells by Southern blot analysis using amplified cDNA from single taste bud cells according to the method described for olfactory neurons (Dulac and Axel, 1995). We obtained cDNA from individual spindle-shaped taste bud cells and used four G probes. However, we noticed that the RNA extraction efficiency and PCR amplification efficiency as estimated from the signal intensity of ß-actin varied during the experiment (data not shown). Therefore, we focused on Gi2, which was found to be expressed in a subset of taste bud cells. We used Gi2-positive cDNA preparations and looked at whether or not the other three G species were expressed in the Gi2-positive cell. As shown in Figure 3, Gi2-positive cells were positive to probes for one to three of the other G species, indicating that multiple G species are co-expressed in each taste bud cell. It was also shown that there is no apparent correlation among the expression patterns of Gs, Gi3 and G_gust; in some cases (nos 7 and 12 in Figure 3) all four G species were expressed and in others (nos 1 and 3) G_gust was not expressed. Further, different cells showed different relative abundance with respect to the expression of the four G species. An example is offered for two cells, no. 15, which is abundant in Gi3, and no. 12, which is abundant in Gi2 and Gs, although these two cells expressed ß-actin equally.

View larger version (70K): [in this window] [in a new window]   Figure 3 Southern blot analysis showing the expression of Gs, Gi2, Gi3 and Ggust in single taste bud cells. Amplified cDNA samples from 16 single taste bud cells positive to the Gi2 probe were electrophoresed, blotted and hybridized at 68°C with radiolabeled probes for Gs, Gi2, Gi3, Ggust and ß-actin. Lane 14 shows the result with amplified cDNA prepared without using reverse transcriptase as a negative control. Some lanes have plural bands with different sizes. This phenomenon is apparently due to the steric hindrance of mRNA molecules.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
Here we have newly identified several G species expressed in taste tissues. This study, together with previous papers (McLaughlin et al., 1992, 1994, Kusakabe et al., 1998), demonstrates that 12 G species are expressed in taste tissues. Our study also shows that, among the 12, G_gust, Gs, Gi2 and Gi3 are the dominant species in taste buds; also, in particular, G_gust and Gi2 are expressed in a subset of taste bud cells. Gi2 is thus expected to play a role in taste signal transduction, as in the case of G_gust. It is also possible that Gi3 and/or Gs, in addition to Gi2 and G_gust, may have physiological functions, such as the tuning of Gi2 and G_gust in taste signaling, because some Gi2-positive cells express Gi3 and/or Gs, while some G_gust-positive cells express Gi3 and/or Gs. On the other hand, the other minor G species, such as G14, G_t-cone and Gi1, may also be of some physiological importance, although their expression levels are lower and/or ubiquitous.

In conclusion, it appears that the composition of G species in taste bud cells is complicated and that multiple G species occur in individual cells. This may be natural in the sense that, within each taste bud cell and between two of them, there are a number of cellular events other than the taste signaling, including cell-to-cell interaction, cell division, cell differentiation and signaling from gustatory nerves (Lindeman, 1996).

One intriguing observation obtained in this study is the cell-type-specific expression of Gi2, which is known to be involved in the negative regulation of adenylyl cyclase to mitigate cAMP signaling (Taussig et al., 1993). In addition, Gi2-associated Gbg subunits are capable of mediating Ca²⁺ signaling as an activator of PLC-b (Camps et al., 1992), and of changing an ion current as an activator of an inward-rectifying K-channel (Kofuji et al., 1995). Since both Ca²⁺ signaling and ion channel modulation are believed to occur in taste signal transduction (Hwang et al., 1990, Berhardt et al., 1996, Spielman et al., 1996, Ogura et.al., 1997), it is probable that there are some GPCR molecules that function by coupling with Gi2 for taste signaling. Further studies will reveal further details to explain the significance of various G species, especially Gi2, that exist in taste bud cells.

	Acknowledgments

 
We express our thanks to Dr T. Asano, Aichi Human Service Center, for providing anti-Gi2 antibody, and to Dr K. Tanemura, University of Tokyo, for assistance with the immunostaining experiments. This study was supported in part by a grant from the Program for the Promotion of Basic Research Activities for Innovation Biosciences, Japan.

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Accepted March 9, 2000

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