Insights into Taste Transduction and Coding from Molecular

Chapter 2

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Insights into Taste Transduction and Coding from Molecular, Biochemical, and Transgenic Studies Robert F. Margolskee Department of Physiology and Biophysics, Howard Hughes Medical Institute, 1425 Madison Avenue, Box 1677, The Mount Sinai School of Medicine of New York University, New York, NY 10029

This review describes studies in taste transduction from my group during the past decade. We have used biochemistry, molecular cloning and bioinformatics to identify and characterize the receptors, G proteins, ion channels and second messenger effector enzymes that serve as signaling elements in taste receptor cells. Gustducin is a heterotrimeric G protein selectively expressed in a subset of taste receptor cells. Knockout mice lacking gustducin's α subunit display markedly reduced behavioral and nerve responses to both bitter and sweet compounds, implicating this G protein in transduction of both bitter and sweet compounds. Gustducin's α subunit activates a phosphodiesterase, while gustducin's βγ subunits (β3γ13) activate phospholipase C. Trpm5, a taste cell-expressed transient receptor potential channel, has been implicated in bitter responses. T1r3, a novel taste receptor, is the sac gene product and a component of a sweet-responsive taste receptor.

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© 2004 American Chemical Society In Challenges in Taste Chemistry and Biology; Hofmann, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Introduction The sensation of taste is initiated by the interaction of tastants with receptors and ion channels in the apical microvilli of taste receptor cells. This fundamental sense enables organisms to avoid toxins (e.g. bitter plant alkaloids) and find nutrients (e.g. sweet carbohydrates). Psychophysics argues that human taste is comprised of five distinct qualities (sweet, sour, bitter, salty and umami (the taste of glutamate)), which correlate well with taste responses of animals monitored by behavioral and nerve recording. Taste receptor cells are specialized epithelial cells with many neuronal properties including the ability to depolarize and form synapses. Taste receptor cells are typically clustered in groups of -100 within taste buds and synapse via their basolateral aspects with afferent taste nerves. How the vertebrate taste cell responds to a given tastant (signal detection and signal transduction), and how this information is coded and processed (sensory coding) have been the key questions guiding my research for the past decade. In this review I will summarize results obtained in my laboratory during the past ten years by our use of molecular, cellular and whole animal techniques to identify the components of the taste receptor cell and characterize their roles in specifying the peripheral and central taste code.

The Elements of Taste Transduction

Gustducin: a Taste Selective G Protein Gustducin is a heterotrimeric G protein that is selectively expressed in ~25% of taste receptor cells (1,2). Gustducin's α subunit (α-gustducin) is closely related to those of the transducins (80% identical, 90% similar) (1), suggesting that α-gustducin might regulate taste phosphodiesterase (PDE) during taste transduction - analogously to the role of the α-transducins in phototransduction. Indeed, PDEs from bovine taste tissue can be activated in vitro by α-gustducin and a-transducin (3; Bakre et al., submitted). That PDE activation by α-gustducin may be involved in taste transduction is supported by the observation that several bitter compounds elicit a decrease in cyclic nucleotide levels in taste tissue which can be selectively blocked by antibodies against α-gustducin (4). Using native taste receptors from bovine taste tissue we have shown that quinine, naringin, denatonium, strychnine, atropine and several other bitter compounds activate gustducin (Figure 1A) (5,6). The Buck (7) and Ryba/Zuker (8) groups have used molecular cloning to identify the

In Challenges in Taste Chemistry and Biology; Hofmann, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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In Challenges in Taste Chemistry and Biology; Hofmann, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. Sucrose (mM)

C

Denatonium Benzoate (mM)

Sucrose (mM)

Nerve Response

Figure 1. a-Gustducin's role in taste transduction. A. Trypsin assays show that in the presence of native taste receptors G protein a-subunits such as agustducin or a-transducin (shown) can be activated by several bitter compounds (note the 32 kDa fragment diagnostic of a-transducin activation). B. Mean preference ratiosfrom48 hr two-bottle preference tests (consumption of tastant vs. water) show that in comparison to wildtype mice (open circles) agustducin null mice (filled circles) require higher concentrations of denatonium benzoate (bitter) or sucrose (sweet) to show avoidance or preference, respectively. G Summated chorda tympani nerve responses show that in comparison to wildtype mice (open circles) α-gustducin null mice (filled circles) display diminished nerve responses to denatonium benzoate or sucrose. Adapted with permission from references 5 and 11. Copyright 1998, 1996.

Denatonium Benzoate (mM)

Preference Ratio



30 T2r/Trb family of taste receptors. A few members of this family of receptors have been shown to respond to various bitter compounds and to selectively couple to gustducin over other G proteins (9,10% suggesting that many, perhaps even all, of these receptors are bitter-responsive. In vivo results from α-gustducin knockout mice have demonstrated clearly that gustducin plays a key role in taste receptor cell responses to bitter and sweet compounds (11). α-Gustducin knockout mice displayed markedly reduced behavioral and nerve responses to the bitter compounds denatonium benzoate and quinine sulfate, and to the sweet compounds sucrose and SC45647 (Figure 1BC). More extensive analysis of taste responses of α-gustducin knockout mice, α-transducin knockouts and α-transducin/a-gustducin double knockouts have shown that α-gustducin is essential to responses to most bitter, sweet and umami compounds, while α-transducin is only involved in taste responses to umami (Danilova, V., He, W., Glendinning, J., Hellekant, G., Damak, S., Margolskee, R. F., unpublished). Transgenic expression of a dominant-negative mutant of α-gustducin inhibited the residual sweet and bitter responsiveness of α-gustducin knockout mice, arguing that another taste-expressed G protein coupled to taste receptors mediated this effect (12). Analysis of a-transducin/agustducin double knockouts indicates that α-transducin does not mediate these residual responses.

Gyl3: Gustducin's γ-subunit We used single cell reverse transcription-polymerase chain reaction (RTPCR) and differential screening to clone cDNAs specific to a-gustducin-positive taste receptor cells (13). Of 40,000 plaques screened, 60 clones were differentially positive when probed with cDNA probes from a-gustducinpositive vs. a-gustducin-negative cells. Two of these clones contained an open readingframeof 201 base pairs, predicted to encode a novel 67 amino acid long G protein γ-subunit, named Gyl3. Gyl3 is only distantly related to the known G protein γ-subunits (25-33% identity). By northern blot we detected a Gyl3 transcript of 0.5 kb in brain, retina, olfactory epithelium, and to a lesser extent, in stomach and testis (Figure 2A). The greatest labeling was seen in olfactory epithelium and cerebellum, followed by retina. In situ hybridization with taste bud-containing tissue showed that Gyl3 mRNA was selectively expressed in taste receptor cells, but absent from the surrounding lingual epithelium, muscle or connective tissue (13). We used "expression profiling" with taste budcontaining tissue and control non-gustatory lingual epithelia to determine that agustducin, G$3 and Gyl3 were only expressed in taste bud-containing tissue, while Gpi was expressed in both gustatory and non-gustatory lingual epithelia (Figure 2B). Profiling the pattern of expression of individual taste receptor cells showed that 19 of 19 cells that expressed α-gustducin also expressed Gyl3 and β3 (Figure 2B).



31 We used three different trypsin protection assays to monitor interactions of Gyl3 with other G protein subunits (Figure 2C). In this way we found that Gyl3 interacted in vitro with α-gustducin. Gyl3 also interacted with Gpl and Gp4 to form dimers (Figure 2C). Because of difficulties in expressing Gp3 it was not possible to use the trypsin assay to assess interactions between Gyl3 andGp3. In a third type of trypsin assay we determined that ΰβ1γ13 dimers enhanced the activation of α-gustducin by taste receptor-containing membranes stimulated by the bitter compound denatonium (Figure 2C). The activation of α-gustducin required: 1. taste receptor-containing membranes; 2. the bitter compound denatonium and; 3. a Gy 13-containing βγ dimer. Hence, we conclude that agustducin, ϋ β ΐ and Gyl3 can associate with each other to form a functional heterotrimeric G protein capable of interacting with denatonium-responsive taste receptors. To determine if Gyl3 does indeed function in taste transduction we carried out quench-flow experiments with taste tissue. The addition of bitter denatonium benzoate to murine taste tissue induced the generation of IP to slightly more than twice the basal level (Figure 2D). When the taste tissue was preincubated with antibodies to Gyl3 the addition of denatonium did not increase IP levels appreciably (Figure 2D). Preincubation with antibodies against Gyl or Gy3 did not reduce denatonium-stimulated generation of IP (data not shown). From these results we conclude that βγ subunit pairs containing Ογ13 mediate the denatonium-responsive activation of taste tissue ΡΙΧβ2 to generate IP . This clarifies the previously puzzling observation that many bitter compounds thought to be transduced by gustducin lead to the generation of IP (14,15), yet antibodies directed against α-gustducin did not block this response, although they did block the gustducin-mediated decrease in taste receptor cell cyclic nucleotides (4). Based on our study of ΰγ13 (13) and work from the Breer (16,17) and Spielman (4,14,15) laboratories we can conclude that Gyl3, G$3 and ΡΙΧβ2, mediate the taste receptor cell's EP response to bitter compounds. Thus, heterotrimeric gustducin mediates two responses in taste receptor cells: a decrease in cyclic nucleotides via α-gustducin activation of phosphodiesterase and a rise in IP via βγ-gustducin (Οβ3γι3) activation of PLCp2. 3

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Trpm5: a Taste-Selective Store Operated Channel From the same single taste cell RT-PCR-differential screen which we had used to identify Gyl3 (13) we also identified a partial clone that was selectively expressed in α-gustducin-positive taste cells. The full-length clone contained an open reading frame of 4077 bp predicted to encode a protein of 1158 amino acids. The encoded protein was identified as a novel member of the TRP (Transient Receptor Potential) family of ion channels. This TRP channel was identified independently by others and named Mtrl (18-20) and subsequently renamed Trpm5 (21). In Challenges in Taste Chemistry and Biology; Hofmann, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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In Challenges in Taste Chemistry and Biology; Hofmann, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. 3

Figure 2. Gyl3 's role in taste transduction. A, The northern blot shows that Gyl3 is expressed at high levels in mouse brain, retina and olfactory epithelium. The size of the RNA markers (in kilobases) is indicated in therightmargin. Abbreviations: olf epi, olfactory epithelium; olfbulb, olfactory bulb; BS/SC, brain stem & spinal cord; thai/hyp, thalamus & hypothalamus. B. Expression profiling shows that Gyl3 is selectively expressed in taste cells along with agustducin and Gfi3. Left panel: Southern hybridization to RT-PCR products from mouse taste tissue (T) and control non-taste lingual tissue (N). Right panel: Southern hybridization to RT-PCR productsfrom24 individually amplified taste receptor cells. 3 '-region probesfromα-gustducin (Gust), Gfil, Gfi3, Gyl3, and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) were used to probe the blots. C. Tryptic digestion assays to monitor interactions between Gyl3, α-gustducin and Gβ subunits. Left panel: a-Gustducin is protected (arrow)fromtryptic digestion in the presence of Gyl3 indicating an interaction between α-gustducin and Gyl3. Bottom panel: Gfil and Gfi4 are protected (arrows)fromtryptic digestion in the presence of Gyl3 indicating an interaction between these Gfi-subunits and Gyl3. Right panel: a-Gustducin is activated by denatonium benzoate (Dena) in the presence of taste receptorcontaining membranes and Gpiyl3 (note appearance of the 37 kDa fragement). D. Denatonium-induced IP production in mouse taste tissue is suppressed by anti-Gy!3 antibodies, implicating Gyl3 in mediating this response. (*) Significantly different compared to Dena (p

Insights into Taste Transduction and Coding from Molecular

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