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A Novel Family of Mammalian Taste Receptors

E. Adler, M.A. Hoon, K.L. Mueller, J. Chandrashekar, N.J. Ryba, C.S. Zuker.  2000. Cell. 100: 693-702.

Reviewed here by John McKillop:

Mammalian taste perception and coding involves complex pathways of receptors, signal transduction, and neuronal stimuli which compared with other sensory systems remains relatively
unexplained at the basic cellular level.  Mammals can distinguish between the five basic tastes of sweet, bitter, sour, salty, and umami.  It is well known which areas of the tongue and palate
contain taste buds for each specific taste, but the pathway by which taste perception is processed and transmitted by those cells remains unclear.  From previous data, it was found that
sweet and bitter taste transduction are mediated by a G protein-linked receptor signaling pathway.

In "A Novel Family of Mammalian Taste Receptors", Adler et al. concentrate on the issue of sweet and bitter taste signal detection by G-protein-linked receptors.  They claim to have
identified a novel family of forty to eighty human and rodent G-protein linked receptors expressed in taste receptor cells in the tongue and palate.  From previous data, it was found that
surface cells from the tongue and palate contain receptors which interact with specific molecules which initiate a signaling cascade leading to neurotransmitter release1.  The overall goal of
the authors is to isolate genes for receptors involved in taste signaling.  This information could then be used to mark cells, define signaling pathways, create topographic maps, and finally
to trace the neuronal circuits to find which neurons for which taste connect to where.  This paper by Adler et al. describes the first step of this long process with the isolation and
characterization of a novel family of human and rodent taste receptors.  This family of receptors named T2Rs were found by searching for G-protein linked receptor sequences in genomic
intervals which were linked to bitter perception of taste.

Adler et al. began their characterization of candidate taste receptors called T2R2s with a figure of the predicted amino acid sequences of human, rat, and mouse T2R genes. Seventy such
amino acids sequences are compared in figure 1 as aligned by ClustalW.  The T2R genes were found to lack introns which might interrupt coding regions.  T2Rs also have a short
extracellular N-terminus.  In addition, this figure demonstrates large conservation of amino acid sequence among species as there is a high percentage of identical residues. Individual
members of the T2R family exhibit 30%-70% identity.  In addition, their are seven predicted transmembrane domains showed as a black bar above the sequences.  These domains each
correspond with a large patch of identity among residues.  These data are intended to suggest that these novel genes are integral membrane proteins and thus possibly receptor proteins.
Also, the large conservation of residues centered around each of the transmembrane domains is intended to emphasize the importance of these regions as centers for the receptor signal
transduction pathway.

Figure 2 provides  further comparison of T2Rs with cladogram showing that the group is structurally diverse yet each distantly related.  These data sets up a piece of their hypothesis that
there are multiple structurally different receptors which process the same taste perception.  The authors also suggest that the high degree of variability in the T2R family reflects the
necessity for the recognition of numerous structurally different ligands.  The characterization of T2Rs is continued in figure 3 as the genes are localized to three human chromosomes, 5,
12, and 7, and mouse, 15 and 6.  Clustering of genes on chromosomes 12 (human) and 6 (mouse) is also depicted by the data.

Figure's 1-3 provide the necessary structural background information which any characterization requires. These initial figures form an orderly progression of data with which the reader
can form an idea of what kinds of proteins are being addressed in this paper.  The data describing sequence conservation at transmembrane domains and the structural diversity of the
entire family provide an initial suggestion that these proteins are indeed G-protein linked taste receptors.  Once the sequence and structural outline has been made, the authors are then able
to address the more complicated claims made in their summary.
This paper by Adler et al. provides an orderly progression of data where claims are clearly made in the abstract and then clearly substantiated in the same order in the results and

The first claim made is that a novel family of forty to eighty G protein coupled receptors expressed in the tongue and palate have been identified.  This claim is then addressed in the text
with a description of how the estimate of 40-80 proteins in the family was reached.  To arrive at estimates of the gene family size, they used high-throughput human genomic sequence
databases which contain a random sampling of 50% of the genes.   Since they found approximately 50 matching T2R genome sequences, the authors concluded that there should be
approximately 100 T2R genes in the genome.  Next, they corrected for inaccurate databases and for the presence of pseudogenes and came up with a final estimate of 40 to 80 T2R
genes.  Thus, the authors did support and explain their initial claims in the text.

Another claim which the authors made in the summary was that the T2R genes are genetically linked to bitter taste.  This claim was also substantiated in the discussion with a description
of mapping techniques involving behavioral taste-choice assays.  The T2R genes were also linked to bitter taste transduction through recombination studies.  The authors continue to
provide evidence for their further claims that T2Rs are expressed in taste receptor cells and that individual receptor cells can express multiple T2R receptors.  These claims are addressed
in figures 4, 5, and 6.  Figure four sets the stage for figures 5 and 6 as an image of the anatomy of specific tasting tissues in mice.  Figure 5 displays the results of multiple in situ
hybridizations which are intended to show that T2Rs are being expressed in taste receptor cells.  Each of the separate tasting tissues, fungiform papillae, foliate papillae, circumvallate
papilla, geschmackstreifen and epiglottis were probed with five different previously tested antisense cRNA probes from five T2Rs.  Panels A through F displays fungiform and foliate
tissue expressing different T2R receptors.  The authors approximate 15% cell expression of T2R in foliate and circumvallate cells. The purpose of this figure is to demonstrate that T2Rs
are being selectively expressed in specific tasting tissues.  It is shown that foliate papillae and circumvallate papilla have more T2R expression than the fungiform, geschmackstreifen and
epiglottis tissue whose expression was clustered and less than 10%.  These in situ hybridizations provide further characterization of the T2R receptors through a connection of location
and function.  This is important evidence as it allows the authors to say thus far that our protein family of interest is being expressed in tissues related to tasting tissues.  This must be
substantiated later, however, with functional assays.

With the knowledge of what percent of tasting cells in specific tissue express T2R, Adler et al. next addressed the issue of coexpression of T2R receptors in individual cells. Figure 6
responds to this question with another series of in situ hybridizations.  Panels A, B, and C, represent circumvallate cells which contain 2, 5, or 10, different T2R probes respectively.  The
T2R expression, however, remained relatively equal in each panel.  The authors were then able to suggest that only a certain specific group of cells express T2R but that those cells which
do express T2R perform coexpression of different T2R receptors.  To test this, panel D was included which demonstrated that coexpression of different T2R receptors does occur.  Thus,
figure 6 further substantiated the author's claims that individual taste cells can express multiple T2R receptors.

Previous data has shown that bitter and sweet transduction is related to the protein gustducin as gustducin knockout mice had shown a decreased sensitivity to bitter and sweet tastants.
Figure 7 addresses this gustducin issue with  double label fluorescent in situ hybridizations.  This figure demonstrated that T2Rs are expressed in only 1/3 of the gustducin positive
circumvallate cells and 1/10 of the fungiform cells.  These data are inconclusive and the authors enter into speculation about various possibilities which they intend to address in their
accompanying paper2.

The discussion concludes with a good summary of the basic questions being asked and some possible explanations for the data which they found.  The authors were also very straight
forward in addressing alternate possible explanations from the one they believe to be true.  For example, Adler et al. mentioned that it was possible that there was expression of T2R at
levels below their ability to detect. Thus, they admit that they could have missed something.  This paper was also part of an orderly progression of data where the authors could first
characterize a novel group of protein receptors and then proceed in the accompanying paper with functional assays of specific T2Rs.  Thus, they provide good background information
before they attempt to narrow in on any one receptor.

In general, the figures were very informative and well placed.  However, the in situ hyrbidation figures could have used some negative controls to show what the background levels were.
Also, how does the reader know that the T2R receptors are not expressed in the majority of the tissues in a human or a mouse.  The authors should have included non-tasting tissue
samples in the experiments to show that T2R is mainly localized in the tongue and palate region.

While the accompanying paper begins a functional assay of a few specific T2Rs, the characterization should have also included a more specific description of individual T2Rs. The
characterization of T2Rs would have been more thorough with specific properties of the receptors.  Also, the authors seemed to concentrate completely on the T2R proteins.  Some
information about levels of mRNA transcription or the process by which the proteins become integral would have been more informative.  This leads into some future experiments which
could be done or perhaps should have been done to provide a more thorough characterization of the T2R family.

Instead of concentrating completely on the T2Rs as proteins, the authors could have done a PCR analysis to show banding patterns.  Also, to explore the expression levels of specific
T2Rs, the authors could have performed RT-PCRs to see which T2Rs were being expressed at what levels.  They could have then compared mRNA production to the in situ hybridization
results to see how mRNA production relates to the amount of completed receptors.  Another useful piece of information would be to know which tissues of the organism were expressing
T2Rs.  The Beta galactosidase expression system could have been used as a reporter such that cells which express a specific T2R receptor would also be blue.  This process, however time
consuming, would allow the tracing of T2R expression to specific tissues.

The final most important future series of experiments are functional assays.  It is important to functionally show that T2R are connected to bitter and sweet taste perception.  This is
precisely what the accompanying paper attempted.  Hoon et al. used a heterologous expression system to associate specific T2Rs with bitter taste perception.  Another functional analysis
could also be to eliminate sections of a T2R gene, one at a time at the genetic level, and trace which parts of the gene sequence can be shown to be necessary or sufficient for a functional
receptor to be made.

Overall, this paper by Adler et al. was informative, well organized, and substantiated their claims.  They chose specific characterization methods which were important for what they wanted
to accomplish, and they set the stage for the accompanying paper.  The authors were self critical, provided explanations for their data, and proposed future experiments which could
advance the knowledge of T2Rs further.


1. E. Adler, M.A. Hoon, K.L. Mueller, J. Chandrashekar, N.J. Ryba, C.S. Zuker.  2000. Cell. 100: 693-702.
2. J. Chandrashekar, K.L. Mueller, M.A. Hoon, E. Adler, L. Feng, W. Guo, C.S. Zuker.  2000. Cell. 100: 703711.

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