First, we used end-point RT-PCR to evaluate the expression of Gαq family subunits in taste buds to assess which members, if any, of the Gαq family (Gαq, Gα11 and Gα14) are expressed in taste buds. We analyzed taste buds obtained from four different oral taste fields (vallate, foliate, fungiform and palate) as well as non-taste lingual and palatal epithelium.

As shown in Fig. 1A, Gα14 is strongly expressed in vallate and foliate taste buds, with somewhat lower expression in the palate. Under parallel conditions, expression of Gα14 was negligible in the fungiform field. Gαq also was not detected in fungiform papillae, and its mRNA was seen in taste buds from vallate, foliate and palate. Finally, we observed that Gα11 appeared to be expressed similarly in taste buds of all taste fields. Of these three Gα subunits, Gαq and Gα11 were detected in non-taste mRNA at roughly similar levels as in taste buds. In contrast, Gα14 was expressed in a highly taste-selective manner. We also tested expression of the distantly related subunit, Gα15. We detected RT-PCR product for Gα15 prominently in the nontaste epithelium samples and very little in vallate taste bud samples (data not shown). Thus, we did not investigate this subunit further. The taste selective expression pattern and apparently high mRNA level of Gα14 suggested that it may be the principal Gαq family member playing a taste-specific role in murine taste buds.

To test this, we undertook quantitative RT-PCR experiments to compare expression levels of the Gαq family members in taste buds (Fig. 1B). The concentration of Gα14 mRNA in vallate taste buds was comparable to that of PLCβ2 mRNA. Neither mRNA was expressed in nontaste epithelium. In contrast, Gαq mRNA was found at similar concentration in CV taste buds and in nontaste epithelium while Gα11 was at much higher concentration in nontaste epithelium than in taste epithelium. Thus, neither Gα11 or Gαq are expressed in a taste-selective manner. Further, the mRNAs for Gαq and Gα11 are expressed at 14- and 80-fold lower concentrations respectively than Gα14 mRNA. These data support our interpretation from end-point RT-PCR, that the only Gα subunit of this family that is likely to have a taste-selective role is Gα14.

Taste buds contain two cell types that have been functionally defined to date, Type II/Receptor cells and Type III/Presynaptic cells. To assess whether Gαq, Gα11 or Gα14 are selectively expressed in these two cell types, we used PLCβ2-GFP and GAD-GFP transgenic mice that respectively illuminate Type II and III taste cells 3334. Individual GFP-labeled cells from each strain were harvested to produce 3 pools, each of 10 cells, representing Type II and Type III cells respectively. We examined expression of the Gα subunits by RT-PCR on amplified RNA from these 6 pools (Fig. 2). Gα14 expression was limited to Type II cells and was detected in each of the three pools. In contrast, Gαq expression was less prominent, and was found in Type II, Type III and non-taste epithelial cells, consistent with the qRT-PCR data of Fig. 1B. Gα11 was only sporadically detected in the pools of identified taste cells, consistent with the low level seen in qRT-PCR with whole vallate taste buds. The absence of Gα11 in the isolated non-taste cells may reflect the heterogeneity of the non-taste epithelium, with some regions expressing Gα11 and others not. In summary, RT-PCR analyses suggested that Gα14, expressed only in taste buds, and only in Type II/Receptor cells within taste buds, was a reasonable candidate for coupling to a taste GPCR.

Next, we used an antiserum that recognizes Gαq, Gα11 and Gα14 to perform fluorescent immunocytochemistry on cryosections of taste epithelia. The Gq/11/14 antibody reacted with a subset of taste cells in posterior (vallate and foliate) but not anterior (fungiform and palate) oral taste fields (Fig. 3). Immunoreactive cells in each case were elongate and spindle-shaped, as is typical for mature taste cells. Immunoreactivity appeared to be membrane-associated, largely encircling the taste cell profiles of immunoreactive cells. Relative to the signal in the vallate papilla, immunoreactivity to Gq/11/14 antibody occurred only rarely in cells of taste buds in the soft palate, and was essentially absent in taste buds of the fungiform papillae and pharynx (Fig. 3).

To assess whether the Gαq-family immunoreactivity was attributable to Gαq, we examined immunoreactivity with the same Gq/11/14 antibody in Gαq-KO mice. In vallate taste buds, staining with anti-Gq/11/14 was essentially no different in Gαq-KO mice compared to WT controls (Fig. 4A, A'). Staining with anti-Gαgus antibody also shows no difference in the Gαq-KO compared to WT animals (Fig. 4B, B') indicating that the Gαq-KO did not disrupt expression of other taste-related G-proteins. The robust staining by the Gq/11/14 antibody in Gαq-KO mice indicates that the bulk of the staining observed with Gq/11/14 antibody must be attributable to Gα11 or Gα14. Because the RT-PCR data (see Figs. 1 &2) showed that Gα11 is expressed at very low levels, the combined analyses suggest that Gα14 is the principal Gαq family subunit in Type II (Receptor) cells.

To further test whether Gα11 is present in taste buds, we utilized an antiserum directed against the N-terminal region of Gα11 which shares no sequence similarity to the N-terminal region of either Gαq or Gα14. This Gα11-specific antiserum does not stain taste buds (Fig. 4C–C') although it does stain the cerebellar molecular layer (Fig. 4E) in which Gα11 is detectable by immunocytochemistry35. These results essentially rule out Gα11 as the source of the taste bud immunoreactivity for the Gq/11/14 antibody. Since this Gq/11/14 antibody exhibits staining in Gαq knockout mice, we conclude that the staining is attributable to neither Gαq nor Gα11 leaving only Gα14 as the possible source of the immunoreactivity. These results are entirely consistent with our RT-PCR data (above) showing that Gα14 mRNA is the predominant Gαq family member isoform expressed in a taste-specific manner.

Taste buds (TBs) comprise at least three different types of mature cells, so we utilized type-specific markers to test whether Gαq-family expression correlates with a specific cell type. Type II (receptor) cells express the GPCR taste receptors (T1Rs and T2Rs in different cells), TrpM5 and PLCβ2 20272829. Thus, we performed the first set of immunocytochemistry using tissues from TrpM5-GFP mice in which all GFP labeled taste cells express the TrpM5 protein28. In vallate taste buds, PLCβ2 antibody stained over 92% (25 of 27 cells) of TrpM5-GFP-labeled cells, confirming the identification of Type II cells with these markers. Further, in vallate taste buds, immunoreactivity to PGP9.5 antibody, which stains mostly Type III cells 26) was detectable only in a small percentage of TrpM5-GFP positive cells (~4%; 1 of 27 cells).

Next, we compared immunostaining for Gq/11/14 with staining for markers of Type II and Type III taste cells. In vallate and foliate taste buds of TrpM5-GFP mice, the Gq/11/14 antibody stained a subset of TrpM5-GFP cells (Fig. 5A). Nearly all Gq/11/14 positive cells expressed TrpM5-GFP (13/14), i.e. Gαq-family protein (presumed Gα14) is expressed only in Type II taste cells. We also immunostained the sections for Gαgus and viewed them for fluorescence in three colors: GFP (green) for TrpM5, Alexa647 (pseudo-colored blue) for Gαgus and Rhodamine RedX (red) for Gq/11/14. Most taste cells that were immunoreactive for Gq/11/14 lacked expression of Gαgus (only 4 of 27 cells co-express these G protein alpha subunits; see Table 1). In contrast, antiserum to PGP9.5 (Type III cell marker) stained none of the Gq/11/14-positive cells (Fig. 5B). We conclude that Gαq-family immunoreactivity is not in Type III cells, but is common in the TrpM5-immunoreactive (Type II) receptor cell population.

Type II cells in taste buds express G protein coupled taste receptors for sweet, bitter or umami qualities. The taste receptors for sweet (T1R2 and T1R3) and for bitter (T2Rs) are expressed in separate subsets of Type II cells18. In taste membranes, bitter (T2R) taste receptors couple functionally to Gαgus 9213637. Yet to date, it is unclear which Gα subunits natively couple to the other class of taste receptors, the T1Rs. Because T1R3 appears to be an obligatory subunit in these dimeric receptors, we used T1R3-GFP transgenic mice, to ask whether the Gαq family subunits are co-expressed with T1R3. By double-immunostaining (Fig. 6), in vallate papillae we found that the Gq/11/14 antibody stained the majority of T1R3-GFP cells (85.7%; 30 of 35 T1R3-GFP cells; see Table 1). In contrast, a smaller fraction of T1R3-GFP cells were strongly positive for Gαgus (~26.8%; 11 of 41 T1R3-GFP cells; see Table 1) (Fig. 6). Thus Gαgus expression does not correlate with T1R3 expression and therefore cannot be an obligatory partner in taste buds in vallate papillae.

We used transgenic PLCβ2-GFP mice to isolate individual GFP-labeled (Type II/Receptor) cells and performed single cell-RT-PCR as an independent test of the expression pattern of Gαq-family subunits and sweet receptors (T1R2, T1R3). Because Gα11 expression was limited to a low concentration of mRNA (Fig. 1B) and only in a few cells (Fig. 2), we did not include it in this analysis. For this detailed analysis, we collected 21 individual PLCβ2-GFP-expressing (i.e. Type II/Receptor) cells and 2 PLCβ2-GFP-negative cells. As expected, TrpM5 was expressed in all 21 PLCβ2-GFP cells, and was not detected in GFP-negative cells (Fig. 7). The taste receptors, T1R2 and T1R3 were detected in approximately half of GFP-positive cells (10 of 21 cells). All 10 of these T1R2+T1R3-expressing cells also expressed Gα14 (Table 1). In contrast, Gαq was detected in 50%, and Gαgus in only 40% of these T1R2+T1R3-expressing cells. In summary, Gα14 is always found in presumptive sweet-sensing Type II/Receptor cells. Additionally, Gα14 was not detected in the absence of T1R3. Thus, we propose that Gα14 may be the principal Gα subunit coupled to the sweet receptors.

All experiments were performed under protocols approved by the Animal Care and Use Committees of the CU Denver School of Medicine and the University of Miami School of Medicine. We used three strains of adult transgenic mice in which GFP is expressed from the promoters of T1R3, TRPM5, PLCβ2, or GAD1313453. In taste buds from each of these strains, previous studies have shown that all cells expressing the relevant endogenous proteins also express GFP 313334. Gαq-deficient mice and littermate controls were obtained from Satya Kunapuli (Temple University, Philadelphia, PA) 54 with permission from Stefan Offermanns (University of Heidelberg, Heidelberg, Germany). This Gαq-null line was generated by replacement of sequence coding for amino acids 246–297 of Gαq by the neo gene and is described in detail in Cho et al55. The genotype of Gq-null mice was determined by PCR and was confirmed by platelet aggregation assay.

We used a Gq-family antibody that is labelled and sold as anti-Gαq/11 (Santa Cruz Biotechnology; sc# 392) although the manufacturer indicates that it likely will react with Gα14. The antigenic peptide, VFAAVKDTILQLNLKEYNLV, is located near the C-terminus, is 100% identical between Gαq and Gα11 and is 90% identical/100% similar in Gα14 (all sequences from mouse). In contrast, Gα15 is only 45% identical/75% similar in this region. Thus, the antibody likely reacts with Gαq, Gα11 and Gα14, but not Gα15. To test the specificity of this broad Gαq family antibody, we also used an affinity-purified Gα11 specific antiserum (Santa Cruz Biotechnology; sc# 394) directed against an N-terminal peptide (aa 13–29 from mouse sequence) that lacks any sequence similarity to either Gαq or Gα14. The cerebellum was used as a positive control tissue for these antisera since Purkinje cells express both Gαq and Gα11 but not Gα14 3556.

Mice were perfusion-fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Taste tissues (vallate, foliate and fungiform papillae and soft palate) were post-fixed (4°C, 60 min); 14 μm cryosections were washed with phosphate-buffered saline (PBS), blocked with 1% normal goat serum, and incubated with rabbit anti-Gαq-family antibody (1:500); at 4°C overnight. The secondary antiserum used was Rhodamine RedTM-X-conjugated AffiniPure™ Fab Fragment goat anti-Rabbit IgG (H+L) (1:100; Jackson ImmunoResearch Lab.; 111-297-003).

To examine co-expression of the Gαq family with other proteins in taste buds, and to avoid cross reactivity of multiple rabbit primary antibodies with a common secondary antibody, we used the Zenon Rabbit IgG Labeling Kit (Invitrogen, Z25308). For this, each primary antibody other than Gαq/11/14 was pre-conjugated to Alexa Fluor 647 so that it did not require a secondary antibody for visualization. After the binding of the first primary and secondary antibodies was complete, the primary antibody-Zenon647 complex was applied to the slides for 80 min at RT in the dark, washed in PB with 0.2% Triton X-100 and postfixed in PFA/PB. Slides were then coverslipped with Fluormount-G. Omission of primary antibodies (detected with Rhodamine Red-X or Zenon) resulted in no apparent fluorescent signal. The primary antibodies used as Zenon complexes were rabbit anti-PLCβ2 (1:200; Santa Cruz Biotechnology; sc# 206); rabbit anti-Gαgus (1:200: Santa Cruz Biotechnology; sc# 395) and rabbit anti-PGP9.5 (Ubiquitin C-terminal hydrolase-L1; 1:200; AbD Serotec; 7863-0504).

All images were collected with an Olympus Fluoview confocal laser scanning microscope (LSCM) FV300 (Olympus Corporation). For each image, the channels were collected sequentially with single wavelength excitation and then merged to produce the composite image using the software Fluoview v5.0. This avoids the problem of bleed-through of images resulting from side-band excitation of the fluorochromes. Brightness and contrast were adjusted in Adobe Photoshop 7.0. Images for Fig. 3 are displayed as negatives to permit visualization of faint fluorescent signal. This was accomplished by using the "Invert" command in Photoshop on the Red channel of the confocal image.

For quantification of immunocytochemical data, we counted immunoreactive cells from 3 sections through the vallate papilla from 2 different animals. An immunoreactive profile was included if it had an elongate morphology extending at least half the height of the taste bud and included an obvious nucleus. Cell fragments not including a nuclear profile were not included in the sample.

Adult PLCβ2-GFP and GAD-GFP mice were killed by CO₂ asphyxiation followed by decapitation, the tongue and palate were removed and a protease mixture consisting of 1 mg/ml collagenase, type A, 2.5 mg/ml dispase (both from Roche Products, Indianapolis, IN) and 1 mg/ml trypsin inhibitor (Sigma, St. Louis, MO) in Tyrode buffer was injected. Tyrode buffer consisted of, in mM: 139 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 Hepes, 10 glucose, 10 Na pyruvate, and 5 NaHCO₃; pH 7.2, 318–323 mOsm. Epithelium was peeled from underlying tissue after 20 min and incubated in Ca/Mg-free Tyrode's solution for 14 min. For Ca-Mg-free Tyrode buffer, CaCl₂ and MgCl₂ were replaced with 2 mM each EGTA and BAPTA.

To avoid contamination of samples with non-taste cells, taste buds from vallate, foliate, fungiform papillae and palate were collected in two stages. First, they were extracted under a stereomicroscope from the epithelium using glass pipettes (inner diameter at tip, 80 μm) and transferred into Tyrode buffer. Next, individual taste buds were identified by GFP fluorescence under under 200× magnification and transferred to lysis buffer (Absolutely Nanoprep kit, Stratagene). Total RNA was purified with DNAse I digestion and first strand cDNA synthesized with Superscript III (Invitrogen, Carlsbad, CA). PCR was performed in 20 μl using cDNA of one taste bud as template per reaction. Positive and negative controls were run in parallel from master mixes. We designed PCR primers using published cDNA sequences for each gene. Because Gαq, α11 and α14 are highly homologous, we placed primers in the most divergent regions. Primers are listed in Table 2. PCR was performed for 35 cycles. Most primer pairs spanned at least one intron to avoid amplifying genomic DNA. PCR products for all genes were validated by DNA sequencing.

Vallate taste buds were collected from PLCβ2-GFP mice (as above), then cells were dissociated by gentle trituration. GFP-labeled single cells were collected each into 60 μl of lysis buffer containing 200 ng of poly-Inosinic acid as a carrier, processed for RNA purification and cDNA synthesis. For preliminary analyses of cell type specific expression (Fig. 2), we collected individual GFP-labeled vallate taste cells, pooled them in lysis buffer and isolated RNA and linear amplified it as previously described 29. The aRNA (amplified RNA) was subjected to reverse transcription and 0.1% of the cDNA was used as template for RT-PCR (40 cycles). For analyses of single cells, mRNA from each cell was reverse transcribed and then each 20 μl single cell cDNA was divided as follows: 5% each for β-actin and TrpM5, 10% each for Gαgus and Gα14, 20% each for T1R2 and T1R3 and 30% for Gαq. PCR was performed in 20 μl for 40 cycles.

Quantitative RT-PCR was carried out as previously described57 using the same primers as for end point PCR, and SYBR Green PCR mix (Bio-Rad) in a Bio-Rad iQ iCycler. Three independent samples of CV taste buds and of adjacent non-taste epithelium were purified and analyzed in parallel. The concentration of each mRNA was compared to a standard curve generated from a sequence-validated template and calculated using MyiQ software (Bio-Rad). All mRNA concentrations were normalized to β-actin mRNA which was run in parallel.