G protein-coupled receptor (GPCR) signaling pathways play an integral role in cell biology.  Chemosensory information, such as olfaction and taste, is an example of signaling that relies upon GPCR signaling pathways.  Olfaction signaling pathways are more extensively researched and understood than taste reception and signal processing.  It is known that mammals have thousands of different receptors that bind odorants and allow mammals to use diverse codes to signal for many different smells.  There are only five main tastes to distinguish between:  sweet, bitter, sour, salty, and unami.  How the different stimuli are recognized, the specificity of taste cells, and the innervation patterns for the taste cells are all unknown.  The authors of this paper attempt to address these questions related to taste coding, specifically focusing on locating the pathways that sweet and bitter tastes are transmitted.

Gustducin, a G protein a unit, is suspected to be involved with a GPCR in the detection of bitter tastes.  It is found in approximately 30% of taste receptor cells in all taste buds.  Currently, there several known GPCR proteins, two of which are T1R1 and T1R2.  Previous experimentation revealed that the T1R family is not expressed with the gustducin and therefore is most likely not responsible for taste transduction for bitter substances.  Careful analysis of the locus 5p15 suspected to be involved in the signal pathway of interest revealed a new GPCR molecule, T2R-1.  Computer investigation revealed a total of 19 additional human receptors in the T2R-1 family.  Figure 1 illustrates the predicted sequences of amino acids for each receptor gene.  Black shaded areas highlight identical residues and gray shaded areas highlight differnces that are due to conservative substituion.  There are seven proposed transmembrane regions indicated by a bar over the corresponding sequence in Figure 1.  The amino acid sequences corresponding to these membrane regions are the most highly conserved areas of the proteins, specifically transmembrane regions 1,2,3, and 7.  The cytoplasmic loops also have high amino acid conservation.  The extracellular domains appear to be the least conserved areas, presumably because this is the domain of the receptor that must bind ligands and therefore need to be variable to recognize many different ligands.  The similarity of the intracellular domains is consistent with the hypothesis that the T2R family of receptors is involved with taste transduction because they should have similar function inside the cell.  The final transmembrane region is located very near the N-terminus leaving a much smaller portion of the protein outside of the cell.  This contrasts with the T1R family, which is characterized by a large extracellular N-terminal domain.  Figure 2 illustrates the phylogenetic relationships between human, mouse, and rat T2R families as well as their distant relationship to vomeronasal receptors and opsins.  The similarity between the receptors ranges from 92% to 46% identity, illustrating the diversity of receptors within the T2R family.

Figure 3 gives a representation of human chromosomes 5, 12, and 7 as well as mouse chromosome 6 and 15.  Using the Genome Sequence Survey database, an estimate of 80 - 120 members in the T2R family was derived.  Considering that a large portion of these T2Rs are psuedogenes pulls the number of functioning T2R GCPRs down by about half, between 40 and 80.  The gray areas on the chromosomes from Figure 3 represent psuedogenes and the red areas represent loci involved with bitter perception.  The gene clusters illustrate the large variety of T2R genes that will encode for diverse taste receptors allowing for wide range of recognition.  Use of radiation hybrid and recombinant mapping studies reveal that the mouse T2R genes are all clustered in only several genomic loci, which are homologous to the genomic loci for human T2R genes.  The loci that contain these homologous genes for mice and humans are all known to deal with bitter taste perception.  In particular, Prp (salivary proline rich protein) is located in the gene clusters of T2R in the bitter regions of the mouse and human chromosomes.  Further evidence that T2Rs are involved in bitter perception was provided by analysis of a DBA/2 x C57BL/g recombinant inbred panel, which showed that the bitter cluster, the receptors and Prp cosegregate.

If T2Rs figure into the taste transduction pathway they must be located in taste bud cells.  Figure 4 is a drawing of the five main regions (Epiglottis, Circumvallate papilla, Foliate papillae, Fungiform papillae, and Geschmackstreifen) containing taste buds and their relative locations within the rat.  In situ hybridization was used to visualize where T2Rs were expressed and to determine if they are located in the taste bud cells.  Probes for the hybridization were constructed from antisense cDNA of five different T2Rs.  Each picture in Figure 5 represents sections from rat Circumvallate papilla that were 1/5 - 1/3 of the taste bud.  Positive results account for 15% of total cells from each taste bud.  The staining is localized in clusters, with the majority of cells expressing T2Rs located in circumvallate papilla (5a - 5e), epiglottis (5h), geschmackstreifen (5g), and foliate (5f) taste buds.   The five different probes all hybridized to the taste cells and would allow each individual taste cell to distinguish between more than one ligand.  Therefore, the location of the T2R receptors complements their projected function in the taste perception signaling pathway.  However, there is a region that T2R does not seem to be highly expressed in, the Fungiform papillae.  Yet, the cells that do express T2R of the fungiform papillae are all clustered closely together (5i) suggesting that there is a common nerve innervating these cells.  One possible explanation for the presence of a few scattered clusters of bitter taste receptors on the tip of the tongue would enable the organism to test a small fragment of food before consumption as opposed to eating a whole bite before realizing the extent of its bitterness.  Thus, the T2R GCRPs are situated in the necessary genomic and physiological locations to be candidates for bitter taste transduction receptors.

To test co-expression of the T2R probes another insitu hybridization was preformed, this time using mixes of different probes.  Figure 6a shows hybridization of 2 probes, 6b shows 5 different probes, and 6c shows 10 different probes.  The number of cells stained when using multiple probes at once was minimally greater than the number of cells stained using one receptor probe at a time, as in Figure 6a - 6c.  However, the intensity of staining when using multiple probes was greater.  One can interpret these data as each cell has a complete set of the receptors, therefore when one probe is used, it will hybridize to the same amount of cells as multiple receptors because they are all in the same cell.  The intensity is brighter because each cell is being stained by more than one probe at a time when multiple probes are used.  Figure 6d directly illustrates the coexpression of two probes, T2R-3 (green) and T2R-7 (red).  Both green and red showed up in the same cells within each taste bud (outlined by the dotted line).

Finally, Figure 7 shows that only T2R is expressed in cells positive for gustducin expression.  In panel a, everywhere that T2R is expressed shows up green.  To the right, in panel b, gustducin is found to be expressed in approximately the same vicinities as T2R.  In order for the gustducin protein to function in conjunction with T2R they must be expressed in the same cells.  Not only are they coexpressed in taste bud cells, but gustducin and T2Rs are also coexpressed in the gastrointestinal tract, trachea, pharynx, nasal respiratory epithelium, ducts of salivary glands, and vomeronasal tissue.  These are all outside of the taste cells and the presence of these two proteins together in these cells as well is a strong indicator that they may function together.  Because gustducin knockout mice are not very capable of detecting bitter and sweet tastants and it seems that T2R is implicitely linked with gustducin,  there is a strong correlation that both T2Rs and gustducin are integrally involved with a bitter taste reception pathway.

The data presented in this article by no means firmly establishes causatory relationships between the T2R family and taste transduction, but it does clearly demonstrate a strong correlation.  Gustducin is a likely part of the signaling pathway for bitter taste reception as evidenced by the decreased sensitivty to sweet and bitter tastes of gustducin knockout mice.  The coexpression of T2Rs with gustducin may be explained by T2Rs’ involvement in recognition of bitter tastants.  Future experimentation in this line of study may yield interesting findings.  There are many questions that this paper raises.  What genes regulate the activity of T2Rs?  Are there mutations in these genes that inhibit expression of T2Rs and what phenotype do these organisms display?  If these genes can be isolated, do they have functions in the transduction of other types of tastants?  Are T2R GCPRs necessary for bitter taste reception, are there any other receptors that gustducin acts in concert with that might be able to replace missing T2R function?  Along this line of study, it would be improtant to determine exactly what role gustducin plays in the signaling pathway.  The earlier mentioned gustducin knockout mice have a decreased sensitivity to various bitter and sweet tastants.  It would be interesting to find out why the deletion of gustducin does not completely erradicate bitter taste reception.  Why are all bitter tastes not recognized?  Why can the knockout mice recognize some bitter tastants and not others?  Are there other G protiens involved in the T2R GCPR signaling pathway?  Can they be isolated and characterized?

Studies on GCPR signaling pathways for other tastants may provide interesting parallels for the bitter taste recognition pathway.  Are there similar proteins involved?  Can the proteins and receptors invovled in bitter taste transduction function in sour taste transduction?  What is the relationship between bitter and sweet taste transduction?  Are there other pairs of tastes that behave in a similar fashion?