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nature neuroscience • volume 2 no 12 • december 1999 1055

Vertebrate taste receptor cells are neuroepithelial cells, organizedinto taste buds of 50 to 150 cells1. Taste receptor cells are ultra-structurally, immunochemically and functionally heterogenous1–3,suggesting that different taste modalities may use distinct mole-cules to transduce sensory signals. Gustducin is a transducin-likeheterotrimeric guanine nucleotide binding protein (G protein)that is selectively expressed in a subset of electrolucent (‘light’)taste receptor cells in the lingual epithelium4,5. However, gust-ducin’s βγcomponents have not been identified or characterized.

Although a number of seven transmembrane-helix receptorshave been indentified in taste tissue and/or taste cells6-9, with theexception of monosodium glutamate and the mGluR4 receptor,no ligands have been identified for these candidate taste recep-tors. In biochemical studies, taste membranes activate gustducinand/or transducin in the presence of any of several bitter com-pounds10,11, demonstrating that presumptive bitter-responsivereceptors in taste membranes may couple to these G proteins.Mice lacking α-gustducin show markedly reduced behavioraland electrophysiological responses to compounds that humansconsider bitter or sweet, implicating the gustducin heterotrimeras a key element in bitter and sweet transduction in vivo12. Cur-rent models of the transduction of responses to bitter compoundspropose that α-gustducin acts via PDE to regulate cyclicnucleotide levels of taste cells, whereas other G-protein α sub-units (α14, α15, αq) and/or Gβγ subunits act via PLC to regu-late levels of IP3 and diacylglycerol (DAG)12–14.

G-protein α and βγ subunits separately and jointly regulatethe activity of many types of ion channels and effector enzymessuch as adenylyl cyclases and PLCs15,16. Gβγ dimers also con-tribute to the specificity of receptor–G protein coupling and to

the regulation of receptor phosphorylation and desensitiza-tion17–21. Five Gβ and eleven Gγ subunits have been isolated frommammals15,22–26. Whereas Gβ subunits share ∼ 50–90% aminoacid identity, Gγ subunits are much more diverse, displaying27–76% identity. As with certain Gα subunits, the expression ofsome Gγ subunits is highly restricted25,26. The functional inter-action of Gγ subunits is selective, but not limited to a single typeof Gα or Gβ subunit. The specificity of subunit interactions with-in heterotrimers, as well as the particular Gγ subunit expressed,are key factors in determining the coupling of receptors to par-ticular intracellular signal transduction pathways.

To gain further insight into the gustducin-mediated mecha-nisms underlying taste transduction, we set out to identify andclone those taste transduction components (receptors, effectorsand Gβ and Gγ subunits) that are selectively co-expressed withα-gustducin in taste receptor cells. In this report, we describe aGγ subunit (Gγ13) that colocalized with α-gustducin in tastereceptor cells. Gγ13 also interacted functionally with α-gustducin,promoted the interaction of α-gustducin with taste receptors andmediated denatonium-responsive activation of taste tissue PLC.

RESULTSTo clone cDNAs specific to subtypes of taste receptor cells, we car-ried out reverse transcription-polymerase chain reaction (RT-PCR)on single taste receptor cells, followed by differential screening ofcDNA libraries from single taste cells27,28. Individual taste recep-tor cells were isolated by limited enzymatic dispersal of circum-vallate papillae29 from transgenic mice that expressed a greenfluorescent protein (GFP) transgene from the gustducin promot-er30. Live GFP-positive taste receptor cells were identified by their

Gγ13 colocalizes with gustducin intaste receptor cells and mediatesIP3 responses to bitter denatonium

Liquan Huang1,2, Y. Gopi Shanker1,2, Jolanta Dubauskaite1,2, Jenny Z. Zheng1,2,3, Wentao Yan4,Sophia Rosenzweig4, Andrew I. Spielman4, Marianna Max2 and Robert F. Margolskee1,2

1 Howard Hughes Medical Institute and 2Department of Physiology and Biophysics, Mount Sinai School of Medicine of New York University, Box 1677, One GustaveL. Levy Place, New York, New York 10029, USA

3 Present address: Regeneron Pharmaceuticals, Inc., 777 Old Sawmill River Road, Tarrytown, New York 10591, USA4 Basic Science Division, New York University College of Dentistry, 345 East 24th Street, New York, New York 10010, USA

The first two authors contributed equally to this work.

Correspondence should be addressed to R.F.M. ([email protected])

Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The α subunit ofgustducin (α-gustducin) is critical for transduction of responses to bitter or sweet compounds. Weidentified a G-protein γ subunit (Gγ13) that colocalized with α-gustducin in taste receptor cells. Of19 α-gustducin/Gγ13-positive taste receptor cells profiled, all expressed the G protein β3 subunit(Gβ3); ∼ 80% also expressed Gβ1. Gustducin heterotrimers (α-gustducin/Gβ1/Gγ13) were activatedby taste cell membranes plus bitter denatonium. Antibodies against Gγ13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducinheterotrimers transduce responses to bitter and sweet compounds via α-gustducin’s regulation ofphosphodiesterase (PDE) and Gβγ’s activation of phospholipase C (PLC).

© 1999 Nature America Inc. • http://neurosci.nature.com©

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1056 nature neuroscience • volume 2 no 12 • december 1999

green fluorescence, and live GFP-negative taste receptor cells werepicked according to their bipolar morphology and lack of greenfluorescence. Messenger RNA from each taste cell was reverse tran-scribed into cDNA, which was then amplified by PCR27,28. PCRproducts were used for target DNA (for profiling gene expression),single-cell cDNA library construction and generation of probeswith which to differentially screen single-cell cDNA libraries.

The cDNA library from an α-gustducin-expressing cell wasscreened with ‘self probe’ (PCR products from the same cell) and‘non-self probe’ (PCR products from an α-gustducin-negative tastecell). Of 40,000 plaques screened, 60 clones were self-probe positivebut negative with the non-self probe. An open reading frame of201 bp, predicted to encode a protein of 67 amino acids homolo-gous to known G protein γ subunits (Fig. 1), was found in 2 ofthese clones. A search of DNA-sequence databases identified twopartially sequenced EST clones from cDNA libraries of humanbrain and rat mixed organs. The rat EST clone (accession numberAI454466) and the re-sequenced human EST clone (accessionnumber H46116) encoded Gγ subunits with 98.5% and 95.5%amino acid identity, respectively, to the mouse Gγ clone (Fig. 1).

Following the convention for naming Gγ subunits, this clone wasdesignated Gγ13. The predicted Gγ13 protein is smaller in lengththan all other known γsubunits, with a calculated molecular weightof 7.9 kDa. Alignment of Gγ13 with other Gγ subunits revealedthat it is the most divergent member of the Gγ family, with great-est identity to Gγ8cone (33% amino-acid identity; Table 1).

Searching databases of human genomic DNA identified aclone from the telomeric region of the short arm of human chro-mosome 16 (16P13.3; accession number AL031033) that con-tained the Gγ13 transcript. The 949-bp human Gγ13 cDNA wasdistributed over 2.6 kb of genomic DNA. Like other Gγ genes,Gγ13 is composed of three exons and two introns22,31–33. The firstexon contains only the 5′ flanking region, the second exon con-tains the translation initiation site and the codons for the first 33amino acids, and the third exon contains the rest of the codingsequence and the 3′ flanking region. The second intron of Gγ13is shifted seven amino acids toward the C terminus with respectto Gγ genes γ1, γ4, γ5 and γ8cone22,31–33, in which the secondintron is located precisely two amino-acid residues downstreamof the highly conserved arginine residue (Fig. 1).

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Fig. 1. Comparison of the predicted amino-acidsequence of Gγ13 withthose of other mammalianGγ subunits. Three aminoacids (shown in blue) varyconservatively betweenhuman (hγ13) and mouse(mγ13) Gγ13. The dis-played order of sequencesis arranged according todescending identity toGγ13. Regions of greaterthan 50% identity areboxed in black with whiteor red letters. Gaps wereintroduced to obtain the optimal alignment. The red bars at the top represent the predicted α-helical secondary structure of Gγ13 using the Chou-Fasman and Robson-Garnier methods49,50. The red boxes with residues shown in red represent the α-helical secondary structures of Gγ1 and Gγ2derived from their crystal structures15. The leftmost arrow at the bottom indicates the location of the second intron in the Gγ1, Gγ4, Gγ5 andGγ8cone genes. The rightmost arrow indicates the position of the intron in the Gγ13 gene.

Table 1. Amino acid relatedness of Gg subunits.

Identity

γ1 γ2 γ3 γ4 γ5 γ7 γ8cone γ8olf γ10 γ11 γ12 γ13γ1 * 33% 30% 31% 27% 38% 62% 31% 31% 73% 34% 25%γ2 48% * 75% 75% 48% 67% 38% 70% 52% 32% 62% 32%γ3 46% 87% * 68% 46% 58% 33% 56% 51% 28% 56% 28%γ4 50% 92% 79% * 43% 56% 31% 63% 46% 30% 53% 32%γ5 46% 76% 71% 73% * 49% 28% 45% 53% 26% 44% 24%γ7 56% 85% 79% 84% 79% * 40% 56% 53% 38% 76% 30%γ8cone 86% 48% 48% 48% 48% 49% * 31% 36% 64% 38% 33%γ8olf 50% 89% 79% 84% 69% 79% 58% * 51% 30% 49% 29%γ10 53% 71% 71% 68% 79% 76% 48% 62% * 31% 41% 24%γ11 90% 48% 48% 49% 48% 59% 78% 51% 54% * 36% 31%γ12 54% 76% 76% 76% 76% 91% 49% 80% 69% 57% * 27%γ13 49% 46% 46% 48% 39% 45% 46% 48% 40% 51% 49% *

Similarity

The percent identity between subunits is shown above the diagonal (bold type), the percent similarity between subunits is shown below the diagonal (italic type).


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Using RT-PCR, we detected Gγ13 RNA in tissue containingtaste buds, but not in the surrounding epithelial tissue (data notshown; see below for cellular localization). To examine the gen-eral distribution of Gγ13 in non-taste tissues, we analyzed north-ern blots with human or murine RNAs. The human Gγ13 probehybridized predominantly to a 1.2-kb mRNA and, weakly, to a6.2-kb mRNA from brain. The 1.2-kb mRNA was also present atlow levels in small intestine and thymus (Fig. 2a). The murineprobe detected a Gγ13 transcript of 0.5 kb in brain, retina, olfac-tory epithelium and, to a lesser extent, in stomach and testis (Fig.2b). Labeling was greatest in murine olfactory epithelium andcerebellum, followed by retina. RNA transcripts of 2.1 kb and,to a lesser extent, 4.8 kb were also detected in olfactory epitheli-um and cerebellum. Both the human and mouse primary tran-scripts were longer than the isolated cDNAs, suggesting additional5′ untranslated sequences in each mRNA; the higher-molecular-weight transcripts could be unprocessed primary transcripts.

In situ hybridization was used to detect Gγ13 expression in tastereceptor cells; Gγ13 was expressed in taste receptor cells, but absentfrom the surrounding lingual epithelium, muscle or connective tis-sue (Fig. 3a and c). Sense-probe controls showed no nonspecific

hybridization to lingual tissue (Fig. 3b and d). The RNA hybridiza-tion signal for Gγ13 was weaker than that for α-gustducin (Fig. 3e);this may be due, at least in part, to the small size of the Gγ13 probe.

To determine which G protein subunits were expressed in taste-bud-containing tissue, we hybridized probes from the 3′ flankingregion of mouse Gγ13, α-gustducin, Gβ1, β2, β3, β4 and β5cDNAs to the amplified cDNAs from a single circumvallate papil-la or a similar-sized piece of non-gustatory lingual epithelium27,28.By this method, we determined that α-gustducin, Gβ3 and Gγ13were expressed only in taste-bud-containing tissue, whereas Gβ1was expressed in both gustatory and non-gustatory lingual epithe-lia (Fig. 4). Using this profiling method, Gβ2, Gβ4 and Gβ5 cDNAswere not detected in RT-PCR amplified products from either gus-tatory or non-gustatory tissues (data not shown).

We next profiled the pattern of expression of Gγ13, α-gust-ducin and Gβ subunits in individual taste cells; the single-cell RT-PCR products were hybridized with the same set of G-proteinsubunit probes used above (Fig. 4). GFP fluorescence was a strongpredictor of α-gustducin expression: 95% (18 of 19) of the GFP-positive cells also expressed α-gustducin, whereas only 1 in 5 ofthe GFP-negative cells expressed α-gustducin. All of the 19 cells

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Fig. 2. Distribution of Gγ13 mRNA in human andmouse tissues. (a) Autoradiogram of a multiple-tissue northern blot (Clontech) of human RNAshybridized with a human Gγ13 cDNA. Each lanecontained approximately 1 µg poly A+ RNA iso-lated from the twelve different human tissues indi-cated. The Gγ13 probe recognizes a 1.2-kbtranscript abundant in brain, which is present atlow levels in small intestine and thymus. The sizesof the RNA markers are indicated in the left mar-gin. The blot was exposed to film for 36 h. (b) Autoradiogram of a northern blot of mouseRNAs hybridized with mouse Gγ13 cDNA (upperpanel). Each lane contained 25 µg of total RNAisolated from the tissues indicated. A 0.5-kb tran-script was detected in all subregions of mousebrain, as well as in retina, olfactory epitheliumand, to a much lesser extent, in stomach andtestis. The blot was exposed to film for 4 d. Thesame blot was stripped and reprobed with a β-actin cDNA probe (lower panel) and exposed for 1 d. The sizes of the RNA markers are indicated in the right margin. PBL, peripheral blood leuko-cytes; small int, small intestine; sk muscle, skeletal muscle; olf epi, olfactory epithelium; olf bulb, olfactory bulb; BS/SC, brain stem and spinal cord;thal/hyp, thalamus and hypothalamus.

Fig. 3. Photomicrographsof frozen sections of mouse circumvallatepapillae hybridized with[33P]-labeled Gγ13 and α-gustducin RNA probes.(a) Bright-field view ofcross-section of circum-vallate papilla hybridizedto the antisense Gγ13probe. (b) Bright fieldview of circumvallatepapilla, Gγ13 sense probecontrol. Because of thelow intensity of the Gγ13signal, the sections in (a)and (b) were photographed unstained to maximize detection of the hybridization signal. (c) Dark field view of circumvallate papilla hybridized to antisense Gγ13probe and stained with hematoxylin-eosin. (d) Dark field view of circumvallate papilla, Gγ13 sense probe control. (e) Bright field view of circumvallate papillahybridized to antisense α-gustducin probe and stained with hematoxylin-eosin. (f) Bright field view of circumvallate papilla, α-gustducin sense probe control.

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expressing α-gustducin also expressed Gγ13 and β3, and 79% (15of 19) of the α-gustducin/Gγ13 positive cells also expressed Gβ1.Gβ1 was also expressed in three of the five cells that were α-gust-ducin/Gγ13 negative, including one cell that was GFP positive.

To determine if α-gustducin and Gγ13 proteins both local-ized to the same taste receptor cells, we used immunocytochem-istry with antisera to α-gustducin10 and Gγ13 on adjacent thin(3 µm) sections of the lingual epithelium. About one-third oftaste receptor cells in circumvallate (Fig. 5), foliate and fungi-form papillae (data not shown) were positive for either α-gust-ducin (Fig. 5a–d) or Gγ13 (Fig. 5e–h). Longitudinal sections ofthe Gγ13-positive taste cells (Fig. 5e and g) showed the elongat-ed bipolar morphology typical of α-gustducin-positive cells5,10

(Fig. 5a and c). Analysis of adjacent transverse sections indicatedthat the vast majority of Gγ13-positive cells were α-gustducinpositive, and vice versa (Fig. 5b, d, f and h); the few exceptionsseemed to be due to the absence of particular taste cells in adja-cent sections as the section level ascended above the cells’ apicalends. We imaged the osculating faces of two adjacent oblique sec-tions stained with α-gustducin/fluorescein (Fig. 5i) or Gγ13/Cy3(Fig. 5j), then superimposed the images (Fig. 5k) to show fullycoincident expression of these two G-protein subunits. Theseresults are consistent with the expression profiling (Fig. 4) anddemonstrate that α-gustducin and Gγ13 are expressed in the samesubset of taste receptor cells.

The colocalization of Gγ13 with α-gustducin in taste recep-tor cells suggests that they interact to transduce taste-cell respons-es. To test this idea, we used a trypsin protection assay that detectsdirect interaction of G-protein α and γ subunits. Gγ, in theabsence of Gβ, can interact directly with Gα; this interactionseems to dictate which Gβγdimer associates with which Gα sub-unit34,35. Gγ13 protected α-gustducin from tryptic digestion to asignificant extent (Fig. 6a). Gγ1, which interacts with the α-gust-ducin-like rod α-transducin, also seemed to interact with α-gust-ducin, albeit to a lesser extent than Gγ13 (data not shown).Gγ8cone (partner of cone α-transducin) and Gγ5 did not pro-tect α-gustducin from trypsin digestion (data not shown).

The expression of Gβ3 and Gβ1 in 100% and 79%, respec-tively, of the α-gustducin/Gγ13-expressing cells profiled suggeststhat these Gβ subtypes might interact with α-gustducin and Gγ13to form heterotrimers. To determine which Gβ and Gγ13 com-binations were capable of forming dimers, we used anothertrypsin assay: Gβ monomers are cleaved at numerous sites bytrypsin, whereas Gβγ dimers are cleaved at a single site, result-ing in a 26-kDa fragment of the Gβ subunit24,36. When the fiveGβ subunits were individually co-expressed in vitro with Gγ13,

only Gβ1 and Gβ4 formed dimers with Gγ13 that were protect-ed from trypsin digestion (Fig. 6b). The lack of protection of Gβ3by Gγ13 was surprising given their co-localization in taste recep-tor cells. However, this negative result may reflect the inability ofthis in vitro assay to support proper processing of Gβ3, ratherthan a functional lack of dimerization of Gβ3 and Gγ13—others also have failed to demonstrate protection of Gβ3 usingthis assay24,36. The functional association of Gβ4 and Gγ13demonstrated by this assay is not relevant to taste transduction,as Gβ4 is not expressed in taste cells.

Taken together, the results shown in Fig. 6a and b and thecolocalization in taste cells of Gγ13, α-gustducin, Gβ3 and Gβ1suggest that Gγ13 may form heterotrimers with α-gustducin andGβ3 or Gβ1 to transduce taste responses. To test this idea, weemployed a trypsin sensitivity assay previously used in our labo-ratory to monitor the activation of α-gustducin heterotrimersby taste receptors10,11. The addition of Gβ1γ13 but not Gβ2γ2enhanced activation of α-gustducin by taste receptor-containingmembranes stimulated by the bitter compound denatonium (Fig.6c). Gβ1γ13 did not enhance activation of α-gustducin by con-trol, non-taste membranes. Thus α-gustducin activation requiredtaste receptor-containing membranes, denatonium and a Gγ13-containing βγdimer. Hence, we conclude that α-gustducin, Gβ1and Gγ13 can associate with each other to form a functional het-erotrimeric G protein capable of interacting with denatonium-responsive taste receptors. We also assayed enhancement ofα-gustducin activation by the addition of Gβ3γ13, but presum-ably because of technical difficulties (see above), our results wereinconclusive (data not shown). Nevertheless, even in the absenceof in vitro data, the coincident expression of Gβ3 and Gγ13 withα-gustducin in the taste receptor cells profiled (Fig. 4) stronglysuggests that they form a heterotrimer in vivo. It is possible thatboth α-gustducin/Gβ1/Gγ13 and α-gustducin/Gβ3/Gγ13 het-erotrimers form in those taste receptor cells that express all fourof these G-protein subunits (15 of 24 cells; Fig. 4). In taste cellsthat express α-gustducin, Gγ13 and Gβ3 but not Gβ1 or anyother known Gβ subunits (4 of 24 cells; Fig. 4), we presume thatthe only heterotrimer that forms is α-gustducin/Gβ3/Gγ13, unlessother, undiscovered Gβ subunits also exist in these cells.

Trypsin sensitivity assays demonstrated that α-gustducin caninteract with Gγ13, that Gβ1 can interact with Gγ13 and that α-gustducin/Gβ1/Gγ13 forms a functional heterotrimer that canbe activated by taste receptors. To determine if Gγ13 functions

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Fig. 4. Pattern of expression of α-gustducin, Gβ1, Gβ3 and Gγ13 intaste tissue and taste cells. Left, Southern hybridization to RT-PCRproducts from murine taste tissue (T) and control non-taste lingual tis-sue (N). Blots were probed with 3′-region probes from α-gustducin(Gust), Gβ1, Gβ3, Gγ13 and glyceraldehyde 3-phosphate dehydroge-nase (G3PDH). Note that α-gustducin, Gβ3 and Gγ13 were allexpressed in taste tissue, but not in non-taste tissue; Gβ1 wasexpressed in both taste and non-taste tissues. Right, Southernhybridization to RT-PCR products from 24 individually amplified tastereceptor cells; 19 were GFP-positive (+), 5 were GFP-negative (–).Expression of α-gustducin, Gβ3 and Gγ13 was fully coincident.Expression of Gβ1 overlapped partially with that of the other G pro-tein subunits. G3PDH served as a positive control to demonstratesuccessful amplification of products. Gβ2, Gβ4 and Gβ5 probes didnot hybridize to the amplified cDNAs from the 24 individual cells or tothe taste or non-taste cDNAs (data not shown).


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in taste transduction, we carried out rapid-time-course quench-flow experiments37. In such experiments, denatonium and sucroseocta-acetate cause the rapid (50–200 millisecond) generation ofIP3 in murine taste tissue, but not in control, non-taste tissue38.Furthermore, the denatonium response depends upon a PLC β2isotype specifically expressed in taste receptor cells14. The additionof denatonium benzoate to murine taste tissue induced the gen-eration of IP3 to slightly more than twice the basal level (Fig. 7),comparable to previous results14,38. IP3 generation was not affect-ed by the addition of either buffer or antibody alone (data notshown). However, when the taste tissue was preincubated witheither of two antibodies to Gγ13 (anti-Gγ13-B or anti-Gγ13-C)the addition of denatonium did not appreciably increase IP3 lev-els (Fig. 7). In contrast, preincubation of the taste tissue withnormal IgG did not reduce the denatonium-stimulated genera-tion of IP3. Likewise, preincubation with antibodies against Gγ1or Gγ3 did not reduce denatonium-stimulated generation of IP3

(data not shown). From these results, we conclude that βγsubunit

pairs containing Gγ13 mediate the denatonium-responsive acti-vation of PLC β2 to generate IP3 in taste tissue.

DISCUSSIONUsing differential hybridization to screen for molecules involvedin taste transduction, we identified Gγ13, a G-protein γ subunitdistantly related to other Gγ subunits. Gγ13 mRNA was expressedin taste cells, retina, olfactory epithelium and several brainregions, but was generally absent from those peripheral tissuesexamined. Gγ13 mRNA and protein were selectively expressedin the α-gustducin-positive taste receptor cells. The concordantexpression of α-gustducin and Gγ13 in taste cells and demon-stration of their physical interaction strongly suggests that Gγ13is the Gγpartner for α-gustducin in taste receptor cells. In addi-tion, our finding that Gβ3 and Gγ13 were expressed in all α-gust-ducin-positive taste receptor cells profiled suggests that theGβ3γ13 dimer is the predominant βγ partner for α-gustducin.However, extensive overlap in expression of β1, γ13 and α-gust-

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Fig. 5. colocalization of α-gustducin and Gγ13 in taste receptor cells from murine circumvallate papillae. Immunocytochemistry was carried out onadjacent thin (3 µm) sections using rabbit antisera against synthetic peptides from α-gustducin and Gγ13, along with fluorescein- or Cy3-conjugatedanti-rabbit secondary antibodies, respectively. Upper panels show α-gustducin immunofluorescence in longitudinal section (a; 200×), transverse sec-tion (b; 100×), longitudinal section (c; 1000×) and transverse section (d; 1000×). Gγ13 immunofluorescence in sections adjacent to those shownimmediately above in the upper panels is shown in longitudinal section (e; 200×), transverse section (f; 100×), longitudinal section (g; 1000×) andtransverse section (h; 1000×). Lower panels show α-gustducin (i) and Gγ13 (j) immunofluorescence in osculating faces of adjacent oblique sections(1000×). (k) Superimposition of two images (in i and j) shows colocalization of Gγ13 and α-gustducin in the same taste receptor cells.


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ducin and demonstration of their physical interaction in ourfunctional assays suggest that α-gustducin/Gβ1/Gγ13 het-erotrimers also form in taste receptor cells. Gγ13’s role and α−subunit partner(s) in retina, olfactory epithelium and brain arepresently unknown.

Identifying the βγ subunits expressed in taste receptor cells,particularly the βγ partners of α-gustducin, is important tofurther our understanding of taste transduction. Previous workfocused on the role of α-gustducin, demonstrating that pre-sumptive receptors present in bovine taste membranes acti-vated α-gustducin and/or α-transducin in response to severalbitter compounds (for instance, denatonium, quinine, strych-nine or nicotine), but not in response to salty, sour or sweetstimuli10,11. Other data implicate α-gustducin and α-transducinin the activation of a PDE expressed in bovine taste tissue10,suggesting that cAMP or cGMP (or both) act as second mes-sengers in transducing responses to bitter compounds.Decreased cAMP/cGMP levels could activate cNMP-inhibitedchannels39, leading to depolarization, or inhibit cNMP-gated

channels40, leading to hyperpolarization. Alternatively (or addi-tionally), α-gustducin/PDE-regulated levels of cNMPs may actvia kinases to regulate the activity of K+ channels via their phos-phorylation state41.

Gustducin’s βγ component(s) might regulate one or moresecond messenger effectors: adenylyl cyclase is thought to beinvolved in responses to sweet taste2,41,42, and PLC has beenimplicated in both bitter and sweet responses14,38,43–45. Althoughmultiple lines of evidence suggest that responses to many bittercompounds involve transduction via α-gustducin and PDE, PLCand IP3 are also implicated in responses to bitter compounds.Quench flow studies have shown that denatonium and sucroseocta-acetate induce a rapid rise in IP3 levels of murine taste tis-sue38. Components of the phosphoinositol signaling pathwaysuch as the IP3 receptor44 and PLC β214 are expressed in tastecells. PLC β2 is present in the same taste receptor cells thatexpress α-gustducin and Gγ13 (Y.G.S. and R.F.M., unpublishedobservations). Antibodies against PLC β2 block the denatoni-um-induced rise in taste tissue IP3 (ref. 14). In contrast, anti-

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Fig. 7. Denatonium-induced IP3 productionin mouse taste tissue is suppressed by anti-Gγ13 antibodies. Samples were stimulatedwith either buffer alone (Basal), 1 mM dena-tonium alone (Dena) or with 1 mM denato-nium plus various antibodies (normal IgG,Gγ13-B, Gγ13-C), then quenched at 50 ms.In those cases where antibodies were added,murine taste tissue samples were preincu-bated at 5 µg per 30 µg taste tissue with nor-mal IgG (IgG) or with two differentanti-Gγ13 antibodies (Gγ13-B, correspond-ing to aa18–32) and (Gγ13-C, correspondingto aa47–59). Data were normalized to thebasal level (100%) and presented as the mean± s.e. of 7–11 data points. *Significantly dif-ferent compared with Dena (p < 0.05) andDena+IgG (p < 0.005); **significantly differ-ent compared with Dena (p < 0.01) andDena+IgG (p < 0.001)

Fig. 6. Tryptic analysis of interactions among Gγ13,α-gustducin and Gβ subunits. (a) Assay of func-tional interaction between Gγ13 and α-gustducintranslated in vitro. Left lane, in vitro-translated α-gustducin, undigested control. Middle lane, in vitro-translated α-gustducin subjected to trypticdigestion in the absence of Gγ13. Right lane, trypticdigest of the mixture of α-gustducin and Gγ13. Thearrow marks the protected α-gustducin thatdemonstrates the interaction of α-gustducin andGγ13. (b) Assay of functional interaction of Gγ13with Gβ subunits. In vitro-translated Gβ1–5 subunitsalone or in combination with in vitro-translatedGγ13 were subjected to tryptic digestion. Thearrows mark the protected Gβ fragments thatresult from interaction of Gγ13 with Gβ1 and Gβ4.(c) Assay of functional activation of in vitro-trans-lated α-gustducin by taste membranes versus non-taste membranes, in the presence or absence of invitro-translated Gβγ dimers, plus or minus 5 mM denatonium (Dena). The arrows mark the locations of undigested α-gustducin (39 kDa) and trypticfragments derived from GTPγS-bound active (37 kDa) and GDP-bound inactive (25 + 23 kDa) forms of α-gustducin. In the presence of taste mem-branes, the addition of Gβ1γ13, but not Gβ2γ2, shifted a portion of the α-gustducin tryptic fragments from inactive (25 + 23 kDa) to active (37 kDa),indicating specific interactions between α-gustducin and Gβ1γ13 and this heterotrimer with denatonium-stimulated taste receptors.

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bodies against α-gustducin do not block the denatonium-induced IP3 rise, but do block a denatonium-responsive declinein taste tissue levels of cAMP and cGMP (W.Y. et al. Am. Chem.Soc. Abstr. 21, 270, 1999), consistent with α-gustducin’s role ofactivating taste PDE. Multiple lines of evidence implicate thegustducin heterotrimer in transducing responses to many bit-ter compounds10–12: its α subunit stimulates PDE to decreasecNMPs, whereas its βγ components (β1/γ13 and/or β3/γ13)stimulate PLC β2 to generate IP3 and DAG.

Micelacking α-gustducin12 are deficient in behavioral andneurological responses to both bitter and sweet compounds, sug-gesting that α-gustducin is directly or indirectly involved in trans-duction of responses to sweet as well as to bitter. Perhaps selectivepairing of α-gustducin with Gβ1γ13 versus Gβ3γ13 in differentsubsets of taste receptor cells may underlie gustducin’s involve-ment in the transduction of both bitter and sweet responses. Theα-gustducin-null mice may also lack βγsubunits (if they are co-regulated with α-gustducin), or may have free βγ subunits inexcess. In either case, the heterotrimer would not form in theabsence of α-gustducin, and receptor-mediated activation of βγsignaling would be lost along with α-gustducin-mediated sig-nals. To dissect out the α- versus βγ-mediated signals will requirethe generation of transgenic mice expressing selectively mutat-ed forms of α-gustducin deficient in PDE interaction or Gγ13,Gβ1 or Gβ3 mutants deficient in PLC β2 activation.

METHODSGeneration of GFP transgenic mice. The GUS8.4GFP construct wasderived from Gus8.4-lac Z30 by replacing lac Z with red-shifted GFP(BRL). Transgenic mice were generated as described30. Founder animalsand their progeny were screened for the transgene by PCR and South-ern hybridization. GFP expression in taste cells of transgenic mice wasscored by fluorescence microscopy.

Construction and screening of cDNA libraries of single taste receptor cells.The lingual epithelia containing circumvallate papillae were isolated fromthe tongues of GFP-transgenic mice by enzymatic digestion29. Individ-ual taste receptor cells were dissociated from the circumvallate papillaeand identified by their green fluorescence and/or their unique bipolarshape. From isolated single cells, cDNAs were synthesized and amplifiedas described27,28. The PCR products were digested with EcoR I and lig-ated into the λZapII vector and screened by ‘self ’ probe (PCR productsfrom the same cell) and ‘non-self ’ probe (PCR products from anothertaste receptor cell), as described28.

Northern and in situ hybridization. Human and mouse Gγ13 DNAprobes were generated by random primed radiolabeling of the respec-tive cDNA. Northern blots, each lane containing 1 µg of human poly A+RNA (Clontech) or 25 µg of mouse total RNA, were hybridized with theGγ13 probes, washed at high stringency and exposed to film with anintensifying screen at –80°C for the indicated times.

[33P]-labeled RNA probes [Gγ13 (0.2 kb) and gustducin (1 kb)] wereused for in situ hybridization of frozen sections (8 µm) of mouse lingualtissue. Hybridization and washing were as described12. Slides were coat-ed with Kodak NTB-2 nuclear track emulsion and exposed at 4°C for 3weeks and then developed, fixed and stained.

Gene expression profiling. Single taste receptor cell RT-PCR products(5 µl) were fractionated by size on a 1.6% agarose gel and transferredonto a nylon membrane. The expression patterns of the isolated cellswere determined by Southern hybridization28 with 3′-end cDNA probesfor mouse α-gustducin, Gβ1, Gβ2, Gβ3, Gβ4, Gβ5, Gγ13 or G3PDH.Blots were exposed for 5 h at –80°C. Total RNAs from a single circum-vallate papilla and a similar-sized piece of non-gustatory epithelium werealso isolated, reverse transcribed, amplified and analyzed as for the indi-vidual cells.

Immunocytochemistry. Polyclonal antisera against two hemocyanin-conjugated Gγ13 peptides (Gγ13-B, aa18–32; Gγ13-C, aa 47–59) wereraised in rabbits. The α-gustducin antiserum was described10. Frozensections (3 µm) of murine lingual tissue (previously fixed in 4%paraformaldehyde and cryoprotected in 20% sucrose) were blocked in3% BSA, 0.3% Triton X-100, 2% goat serum and 0.1% sodium azide inPBS for 1 h at room temperature and then incubated for 8 h at 4°C withpurified antibody against α-gustducin10 or antiserum against Gγ13(1:1000). The secondary antibodies were Cy3-conjugated goat-anti-rab-bit Ig for Gγ13 and fluorescein-conjugated goat-anti-rabbit Ig for α-gust-ducin. Immunoreactivities of α-gustducin and Gγ13 were blocked bypreincubation of the antisera with the corresponding synthetic peptidesat 50 µM and 1 µM, respectively. Comparable results were obtained witheither anti-Gγ13-B or -C. Preimmune serum did not show anyimmunoreactivity. Adjacent (3 µm) sections were treated with anti-α-gustducin or anti-Gγ13 along with the appropriate secondary antibody.In some cases (Fig. 5i–k), the osculating faces of the adjacent sectionswere imaged, and one of the images was rotated 180° and superimposedwith the other.

Trypsin protection assays. To monitor G-protein α and γ subunit inter-actions35, Gγ13 and α-gustducin were translated in vitro (Promega TNTsystem), mixed in a 3:1 ratio and incubated for 15 min at 30°C in bufferG (15 mM Na-HEPES, 250 mM NaCl, 0.6 mM EDTA, 0.6 mM DTT, 5 mM MgCl2, 0.1 mM GDP and 0.3% polyoxyethylene 10-lauryl ether(LPX) at pH 8.0). The assay was initiated by adding TPCK-treated trypsin(0.5 µg), incubated for 1 h at 30°C and terminated with 3 µg soybeantrypsin inhibitor (SBTI; 15 min at 30°C). The samples were analyzed on12% SDS-PAGE; gels were fixed, enhanced with En3Hance (New Eng-land Nuclear, Boston), dried and exposed to X-ray film. To assay theinteraction of Gγ13 with Gβ subunits24, plasmid DNAs (0.5 µg) for eachof the five Gβ subunits were transcribed and translated in vitro with orwithout 0.5 µg plasmid DNA coding for Gγ13. Aliquots (10 µl) of theco-translated Gβγ mix or Gβ subunits alone were digested by TPCK-treated trypsin (1 µg) in a final volume of 20 µl (with 50 mM Na-HEPES,pH 8.0). After incubation for 1 h at 30°C, the digestions were stoppedby addition of 6 µg SBTI as described above. Protected fragments of Gβsubunits were analyzed on 15% SDS-PAGE. Detection was as describedabove.

To study the role of Gγ13 in the activation of gustducin heterotrimerby stimulated taste receptors, 5-µl aliquots of α-gustducin translated invitro were incubated with 5 µg of purified taste membranes or controlnon-taste membranes, 0.1 mM GDP and 1 µM GTPγS, with or without 5 mM denatonium, and with either a 10-µl aliquot of co-translated Gβ1γ13or Gβ2γ2 dimer or 10 µl rabbit reticulocyte lysate (the minus βγcontrol)for 1 h at 30°C, followed by digestion with trypsin as described10,11.

Quench flow assays. Tongues were excised from 6–8-week-old femaleSWR mice, and lingual papillae removed as described42,46. One vallateand two foliate papillae and nongustatory control tissue from the dor-sal eminence of the peeled epithelium were placed in ice-cold MOPSbuffer (50 mM MOPS, 100 mM NaCl, 0.081 mM CaCl2 and 2.5 mMMgCl2 at pH 6.9), containing 10 mM EGTA, 1mM DTT, and a proteaseinhibitor cocktail (1 mg per ml, specific for serine, cysteine, aspartic andmetallo-proteinases; Sigma). Tissue collected from 25 tongues washomogenized in MOPS-EGTA buffer pH 6.9 without enzyme inhibitorsand centrifuged (1000 × g for 20 min at 4°C). The recovered supernatant(∼ 30 µg per ml protein47), was used for rapid kinetic experiments. Thetissue was prepared fresh and kept at 4°C before the quench-flow exper-iment and loaded into the quench-flow module (QFM) in small batch-es a few seconds before injection. The conditions, set up and operation ofthe QFM were as described37,38. Second-messenger extraction proce-dures were as described48.

For incubations with antibodies, small batches were aliquoted andantibody (or in the case of controls, an equivalent amount of buffer)added to the tissue, then the samples were left on ice to equilibrate (90min). Affinity-purified anti-Gγ13-B and anti-Gγ13-C antibodies, puri-fied normal rabbit IgG and affinity-purified antibodies to Gγ1 and Gγ3subunits (Santa Cruz Biotechnology) were used at identical protein con-centration (5 µg per 30 µg tissue protein) and purity.

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ACKNOWLEDGEMENTSWe thank N. Gautam and D. Logothetis for providing the cDNA clones for Gβ and

Gγ subunits, W. He for help in isolation of taste cells, A. Kozak for help with in-situ

hybridization and T. McClintock, J. Kay, L. Ruiz-Avila, E. Basyuk, L. Briggemann,

R. Ramkumar and B. Knox for discussions. R.F.M. is an Associate Investigator of

the Howard Hughes Medical Institute. This research was supported by NIH grants

DC03155 (R.F.M.), MH57241 (M.M.), DE10754 (A.I.S.) and DC00310 (L.H.)

and by grant M93-14 from the BARD foundation (A.I.S.).

RECEIVED 13 SEPTEMBER; ACCEPTED 30 SEPTEMBER 1999

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