Adler E., Hoon M.A., Mueller K.L., Chandrashekar J, Ryba N.J.P., C.S. Zuker. 2000. A Novel Family of Mammalian Taste Receptors. Cell 100: 693-702.

The mechanism behind the chemosensory perception, also known as taste, in mammals has eluded scientists for quite some time. In a recent paper by Adler et al. (2000), they have identified and isolated a family of proteins responsible for the ability to taste bitter chemicals. As Adler et al. reason, the bitter sensory ability is an precautionary adaptation which serves as a warning device to protect organisms against potentially harmful compounds.

Figure 1 shows a sequence comparison of the proteins translated from different human, rate and mouse T2R genes. The sequences were aligned using ClustalW, and shows that there is a low level of sequence similarity between the sequences, except in those sections that are predicted to be trans-membrane domains. Within these domains, many of the amino acids appear to be conserved, or have conservative substitutions (especially in TM6). Previous work (Hoon et al, 1999) has shown that the proteins produced by these T2R genes are expressed in distinct quantities within taste buds, though their specific function was unknown.

Figure 2 is a cladogram showing the homology between the different T2R receptor proteins between rat, human, and mouse. Some of the T2R genes have intermediate to high homology (mouse T2R-2 and rat T2R-8, 74%; mouse T2R-18 and rat T2R-2, 92%). Between humans and mice, the similarity of several of the T2R's is between 46% (human T2R-1 and mouse T2R-19) to 67% (human T2R-4 and mouse T2R-8).

Figure 3 is a map of the T2R genes to known loci that are associated with bitter taste. Several genes have been found to produce proteins which respond to bitter sensation: Cyx (cyclohexamide), Qui (quinine), Rua (raffinose undeaacetate), and Soa (sucrose octaacetate). This figure shows that these known genes form a bitter cluster, which are then linked to the T2R genes in mice. For humans there is a nine-gene cluster comprised of different T2Rs. Between the different T2R genes, in both mice and humans, there are several T2R pseudogenes which are estimated to make up more than one-third of all the T2R genes.

Figure 4 is a diagram of the inside of the rodent mouth, showing the different tasting regions, all containing taste buds. These different regions will be explored for the presence of T2Rs.

Figure 5 shows the expression of different T2Rs in different cells in the mouse mouth using in situ hybridization with dioxygenin-labeled T2R anti-sense RNA probes. The authors rationalize that if the T2R proteins are in fact responsible for bitter taste, they should be present in taste receptor cells. Figures 5a-e show a high expression of different T2Rs in the rat circumvallate (back of the tongue) taste buds: rT2R-7 (5a), rT2R-8 (5b), rT2R-3 (5c), rT2R-2 (5d), and rT2R-4 (5e). Figure 5f shows the foliate (in front on circumvallate, but still near the base of the tongue) cells expressing rT2R-7, Figure 5g shows the geschmackstreifen (front roof of the mouth) expressing rT2R-3, but in a lower quantity seen than 5a-e and 5f. Figure 5h shows the epiglottis expressing rT2R-7 in the one taste but seen on the tissue section. Figure 5i shows the fungiform (tip of the tongue) with one taste cell positively expressing T2R (though these T2Rs are not specifically described). This figure shows that the rT2Rs are in the highest quantity in taste buds in the circumvallate, traditionally the region of the tongue associated with bitterness.

Figure 6 shows that several different rT2Rs are expressed at the same time within the same cell. Circumvallate cells were probed with a combination of different probes: 2 (6a), 5 (6b), and 10 (6c). In Figure 6d, FISH with different colored probes shows simultaneous coexpression of T2R-3 and T2R-7.

Figure 7 explores the correlation between the expression of gustducin and the expression of T2Rs. FISH with two labels. Figure 7a shows the expression of T2R, and 7b shows that gustducin is also expressed in those same taste cells located in the circumvallate. Figure 7c and 7d are two-channel fluorescent images, with 7c showing some of the gustducin-positive cells do not express T2R. Figure 7d shows that T1Rs and T2Rs are localized with their expression within the taste bud cells.

Evaluation/Commentary

The authors state that they use human T2Rs to screen mouse libraries, which makes sense, but with the limited sequence similarity between the mouse and human T2Rs (Figure 1), some sort of proof that the human T2R sequence binds to/detects mouse T2R would be nice.

The authors propose that they have shown that the T2Rs are expressed in "~15% of the cells of the circumvallate, foliate, and palate taste buds." This statement seems to imply that there is a uniform distribution of the T2Rs within the oral cavity, even though Figure 5 shows that there isn't.

In Figure 5, the authors claim to be able to differentiate which members of the T2R family are expressed in certain localities in the mouth. However, there is no indication of how the authors discriminate between the different T2Rs. Again, it would be nice to see sequence of the different antisense-RNA probes used to see some sort of basis for differentiating between the different T2Rs in the hybridization figure. As we know nothing about the probe, the skeptical reader has no evidence that only T2R RNA and not other mRNA is being labeled within the taste bud cells. I would like to see another negative control of some portion of the oral cavity which is known not to contain taste cells and see it probed for T2R.

In Figure 6, the authors try to show the simultaneous coexpression of different T2Rs. Firstly, there doesn't appear to be too much difference between panels a, b, and c. I see little difference between adding 2 probes and adding 10 probes. I think that panel d illustrates coexpression much better than the other three panels.

In Figure 7, the authors demonstrate that T2Rs are expressed in cells which have gustducin (panels a and b). However, to prove that gustducin is required for T2R expression, I would like to see gustducin-free cells probed for T2R as a negative control. With such an added control, it might be possible to say that gustducin-expression is necessary for T2R expression.

In the context of the theory and previous research (Fig. 1-3) on both T2Rs as well as bitter-reception pathways, the authors make it seem obvious that the T2Rs are responsible for bitter taste. However, the experiments in this paper only seem to conclude that the T2Rs are expressed in specific regions of the oral cavity and taste buds. Only the functional test in accompanying paper from the same lab (Chandrashekar et al, 2000) allows the reader to confirm that the T2R genes are responsible for bitter taste. I think that this paper shows that the T2Rs are associated with taste buds (and probably taste), but do not prove that they are responsible for bitterness.

Future Directions

Even though Adler et al. (2000) and Chandrashekar et al. (2000) demonstrate the role of the T2R genes in bitter taste reception, there is still work to be done.

Specific T2R Function

Discerning the specific bitter perceptions for the different T2R strains remains to be elucidated. Chandrashekar et al. (2000) suggest that mT2R-5 appears to be responsible for cyclohexamide perception. Through creating different T2R knockout mice, each missing different T2R genes, it might be possible to test sensitivity to different bitter compounds via a comparative response. If certain T2R-x minus cells failed to show reception to a certain specific type of bitter compound, this might suggest which chemical compounds can be detected by the missing T2R. Should T2R-x+ genes respond to that same compound, this would implicate that T2R gene as the receptor for that type of compound.

Necessary/Sufficient

While Adler et al. and Chandrashekar et al. show that the T2R genes are involved in bitter perception, they have yet to show what is necessary and what is sufficient for the bitter perception. Since Chandrashekar et al. (2000), suggest that mT2R-5 is responsible for detecting cyclohexamide, this could be used as model for an experiment to determine which domains of the protein are necessary for the T2R detection.

The T2R proteins each have 7 transmembrane domains, and from the topology predicted by Chandrashekar et al. (2000; fig. 6a), there appear to be 3 large extracellular domains. As a taste cell detects external stimuli, it is probably these external domains which play a role in compound detection. By cutting out the DNA coding sequences which produce the extracellular amino-acids; and ligating in a different DNA coding sequence which would produce different extracellular domains, and inserting this new sequence back into a stem cell, it would be possible to create a chimeric mT2R-5 mouse. Using different chimeras, and testing the functional response of the mT2R-5 to cyclohexamide, it might be possible to see which part of this extracellular domain is necessary and sufficient for bitter detection.

Connectivity Patterns

Chandrashekar et al. (2000) state that if one were to label the protein or the cells which express mT2R, the connectivity patterns fo the proteins and cells could be studied. This could be done using green flourescent protein (GFP), and any one of the mT2R genes could be labelled in vivo. Shining flourescent light on the mouse (or plated cells) and illuminating the labelled T2R proteins would allow one to see how the proteins were interacting inter- and intra-cellularly.

Human/Mouse Comparisons

The mT2R genes are providing a wealth of information about bitter taste reception. However because the mouse and human T2R genes can be quite different in structure (and possibly sequence), this mouse model with mT2R might be of limited use to investigate the specific properties of human bitter taste. It would be possible to create a transgenic mouse with human T2Rs and study the functional role of hT2R in vivo. Such a study would shed light on which human genes were comparable to specific mice genes.

The research on bitter-taste pathways might serve as a model for exploring other taste sensations: do the sweet and sour pathways follow a similar mechanism?

Adler E., Hoon M.A., Mueller K.L., Chandrashekar J, Ryba N.J.P., Zuker C.S. 2000. A Novel Family of Mammalian Taste Receptors. Cell 100: 693-702.

Chandrashekar J., Mueller K.L., Hoon M.A., Adler, E., Feng L., Guo W., Zuker C.S., Rybal N.J.P. 2000. T2Rs function as bitter taste receptors. Cell 100: 703-711.

Hoon M.A., Adler E., Lindemeier J., Battey J.F., Ryba N.J.P., Zuker C.S. 1999. Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell 96 541-551.

Wong, G.T., Gannon, K.S., Margolskee, R.F. 1996. Transduction of bitter and sweet taste by gustducin. Nature 381: 796-800.

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