Bitter-Sweet Research

By design, humans crave sweet-tasting foods, which supply necessary calories, and avoid bitter-tasting foods, which could be poisonous. But an individual's genetic makeup can acutely tune taste buds. Visitors to Linda Bartoshuk's Yale University lab can take a simple taste test to discover genetic influences on their food intake. The test measures sensitivity to the chemical 6-n-propyl-thiouracil, which is intensely bitter to acute taste buds, moderately bitter to a medium taste bud, and tasteless to an insensitive bud. Editor's Note: This is the third article in a five-part series on the senses. The next article, on the sense of touch, will appear in the Oct. 29 issue. Sensitive tasters, or supertasters, generally have more taste buds--and they are often women. To them, vegetables are more bitter, fats creamier, and chili peppers hotter. Conversely, nontasters are more likely to eat excessively sweet, very fatty, and highly spiced foods. Not surprisingly, these sensory differences influence food choices, and therefore, health. Those with sensitive taste tend to be thinner because they have more taste buds. Alcoholics are more likely to be insensitive tasters, since they are less repelled by alcohol's bitter taste, Bartoshuk says. "If there's something that we can do to make certain foods more or less attractive to people, this has tremendous dietary significance." The Basics The taste buds occupy the circular-shaped papillae covering the tongue. Each bud holds between 50 and 100 taste cells, which poke through the bud opening. Some cells respond more readily to a distinct taste, such as sweet, bitter, salty, or acidic, or to amino acids such as glutamate. When a taste stimulant, or tastant, interacts with taste proteins (called taste receptors) or with ion channels on the cells' surfaces, electrical changes occur that result in a signal ultimately reaching the brain to register a taste. The chemicals that produce sour and salty tastes act directly through ion channels, while those responsible for sweet and bitter bind to taste receptors, setting off signals that ultimately result in ion channels opening and closing. Until 1992, scientists knew little about how the sense of taste really worked. In that year, Robert F. Margolskee, while at the Roche Institute of Molecular Biology, identified taste transduction components. Margolskee, now a Howard Hughes Medical Institute associate investigator and a professor of physiology and biophysics at the Mount Sinai School of Medicine in New York, identified gustducin, a guanine-binding regulatory protein (G-protein) involved in stimulating the electric transduction of sweet and bitter taste signals. Behavioral studies on mice, altered to lack functioning gustducin, proved the G-protein essential to transducing sweet and bitter tastes.1 This finding stimulated a surge of research on the molecular basis of taste, which was further advanced with the mapping of the mouse and human genomes. Recent research in Margolskee's lab and elsewhere has focused on identifying the taste receptors. Called the taste system's "holy grail" by one investigator, these receptors begin the taste process, providing tools to dissect the system's biology. "By understanding the molecular mechanisms underlying how we taste--the protein targets of the receptors and the signaling cascade downstream of the receptors--we can understand how we detect sweet, sour, salt, and bitter," Margolskee says. "Then we can understand why things go wrong. Each of these types of taste...have importance to human nutritional status." Identifying Bitter and Sweet Genes In 1999, molecular biologist Charles Zuker, University of California, San Diego, and collaborator Nicholas Ryba, at the National Institute of Dental and Craniofacial Research, identified the first two mammalian taste receptors called T1R1 and T1R2 (taste receptor family 1, members 1 and 2). Although they didn't know what taste these receptors responded to, they identified them as G-protein coupled receptors with sequence homologies to other candidate chemosensory receptors.2 The following year, the Zuker and Ryba labs and Linda Buck's lab at Harvard Medical School identified a novel family of receptors, dubbed T2R (taste receptor family 2).3,4 Like T1R1 and T1R2, the T2R genes selectively expressed in taste receptor cells. The Zuker and Ryba groups demonstrated by functional expression that T2Rs coded specifically for bitter tastants.5 The labs found that approximately 25 T2R proteins are part of the bitter taste receptors' large family. Earlier this year, a group of laboratories including the Buck and Margolskee labs identified a candidate sweet receptor gene, called T1R3 (taste receptor family 1, member 3).6-8 The findings grew out of last year's mapping of the mouse Sac locus, a region known to govern the preference for sweet-tasting compounds.9 When Gary Beauchamp, director of the Monell Chemical Senses Center in Philadelphia, first mapped the Sac locus, it was unclear if it was one gene, multiple genes, or a genetic control element, Margolskee says. Beauchamp had pinpointed the Sac locus to a small chromosome region near a known marker, and Margolskee located the homologous marker in humans. Putting together a contiguous human genome sequence, Margolskee then discovered only one gene that coded for a protein, fitting the presumption that the Sac gene product would be a taste cell that expressed a signal transduction component such as a GPCR, he says. It was the sweet taste receptor T1R3.7 The experiments identified the specific amino acid sequence that is probably critical in its function as a sweet receptor. Margolskee characterized the biochemical and pharmacological properties of T1R3 by modeling the amino acid structure of its taster and nontaster form. The amino acid differences affect the receptor's dimerization, and based on homology to the metabotropic glutamate receptor's structure, he predicted dimer formation between two T1R3 units. In the nontaster, the structure of the asparagine N-linked glycosylation site was predicted to interfere with the protein's ability to dimerize. "If that dimerization is important for ligand binding or other functions of the receptor, that could explain the differences between the taster versus the nontaster form of the receptor," Margolskee says. Progress in Functional Courtesy of David V. SmithThis barrel-shaped structure is the mammalian tastebud which contains different cell types: basal, dark and light cells. The Zuker and Ryba group recently confirmed that T1R3 binds sweet ligands. The group completed the transgenic rescue experiments, proving that the Sac locus encodes T1R3. Their experiments also revealed surprising details about how these receptors operate.10 In conducting tests on the sweet receptor, the researchers found that receptors actually combine to detect sweet tastes. Their experiments demonstrated that T1R2 and T1R3 (T1R2+3) combine by dimerization to recognize diverse sweet-tasting molecules such as sucrose, saccharin, and dulcin. "This is quite an unexpected finding," Zuker says. "We know that many G-protein coupled receptors dimerize, but most are homodimers. This is a pretty cool example of a heterodimer." Their experiments indicate that the back of the tongue and palate contain all of the T1R2+3 expressing cells while the front responds to the T1R1+3 expressing cells. Since different nerves innervate the tongue's front and back, the researchers conclude that the two combinations exhibit different connectivity pathways. Zuker and Ryba felt that with the sweet and bitter receptors confirmed, the taste receptors' organization at the periphery could be determined. Their experiments suggest that the cells expressing the bitter T2Rs are distinct from those that express the sweet T1Rs, and that no overlap exists. "These two types of cells mediate such opposite behaviors that it's not surprising," Zuker says. He concedes, however, that such segregation might only exist at the receptor levels, and the signals might mix at the nerve level. Debate: Taste Cell Organization The binding of a taste receptor to its ligand is the initial signaling step in gustatory neuron activation. The long-standing question, however, is whether individual neurons are tuned for one taste (label-lined), or whether taste is population or pattern coded, so that individual cells respond to a spectrum of taste modalities (mixed-lined). The issue has passionate supporters, and data, for both sides. "Despite the Zuker-Ryba group coming down firmly on the side of labeled line and segregation of T1R and T2R taste cells, there is strong evidence from taste cell recording and taste nerve recording to indicate that these cells and fibers are rather more promiscuous and probably use an across-fiber (mixed-line) pattern," Margolskee says. Recent whole-cell electrophysiological experiments and several earlier intracellular electrophysiological experiments support the mixed-line theory. The latest studies by David V. Smith, professor and vice chairman, department of anatomy and neurobiology, University of Maryland School of Medicine, and Timothy A. Gilbertson, professor in the department of biology at Utah State University, recorded the gustatory sensitivities of taste cells in response to taste solution applications like sucrose, salt, and quinine. The recordings showed that while each neuron responds most strongly to one tastant, it typically responds to at least one other tastant with dissimilar taste qualities.11 "We've essentially confirmed that these cells are broadly tuned, but many people are not ready to believe it," Smith says. "We have known from neurophysiological studies for years that sweet and bitter are very independent in the periphery," Bartoshuk says. Organisms must be able to seek out certain tastes and reject others to meet nutrient needs of the body, she argues. The discovery of genes encoding for taste receptors is just the first stage of the processing of taste stimuli, and much can happen between a ligand binding with its receptor and taste perception, researchers note. Says Zuker: "Ultimately, we want to know how the cells are organized in taste buds, how the taste buds are organized on the tongue and innervated, and how the taste signals move upstream to the ganglion and brain taste centers. "We want to understand how the brain knows that sugar is sweet and tonic water is bitter." Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor to The Scientist. References 1. S.K. McLaughlin et al., "Gustducin is a taste-cell-specific G protein closely related to the transducins," Nature, 357:563-9, 1992. 2. M.A. Hoon et al., "Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity," Cell, 96[4]:541-51, 1999. 3. H. Buck et al., "A family of candidate taste receptors in human and mouse," Nature, 404:601-4, 2000. 4. E. Adler et al., "A novel family of mammalian taste receptors," Cell, 100[6]:693-702, 2000. 5. J. Chandrashekar et al, "T2Rs function as bitter taste receptors," Cell, 100[6]:703-11, 2000. 6. J.P. Montmayeur et al., "A candidate taste receptor gene near a sweet taste locus," Nature Neuroscience, 4[5]:49-28, 2001. 7. M. Max et al., "Tas1r3, encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac," Nature Genetics, 28[1]:58-63, 2001. 8. E. Sainz et al., "Identification of a novel member of the T1R family of putative taste receptors," Journal of Neurochemistry, 77[3]:896-903, 2001. 9. X. Li et al., "High-resolution genetic mapping of the saccharin preference locus (Sac) and the putative sweet taste receptor (T1R1) gene (Gpr70) to mouse distal Chromosome 4," Mammalian Genome, 12[1]:13-6, 2001. 10. G. Nelson et al., "Mammalian sweet taste receptors," Cell, 106[3]:381-90, Aug. 10, 2001. 11. T.A. Gilbertson et al., "Distribution of gustatory sensitivities in rat taste cells: whole-cell responses to apical chemical stimulation," Journal of Neuroscience, 21[13]:4931-41, 2001.

Bitter-Sweet Research

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