Vol. Cell, 100, 703711, March 17, 2000, Copyright 2000 by Cell Press
T2Rs Function as Bitter Taste Receptors
Jayaram Chandrashekar,* Ken L. Mueller,* Mark A. Hoon, Elliot Adler, Luxin Feng, Wei Guo,* Charles S. Zuker,* and Nicholas J. P. Ryba * Howard Hughes Medical Institute and Department of Biology Department of Neurosciences University of California, San Diego La Jolla, California 92093 National Institute of Dental and Craniofacial Research National Institutes of Health Bethesda, Maryland 20892 Aurora Biosciences 11010 Torreyana Road La Jolla, California 92121
Summary Bitter taste perception provides animals with critical protection against ingestion of poisonous compounds. In the accompanying paper, we report the characterization of a large family of putative mammalian taste receptors (T2Rs). Here we use a heterologous expression system to show that specific T2Rs function as bitter taste receptors. A mouse T2R (mT2R-5) responds to the bitter tastant cycloheximide, and a human and a mouse receptor (hT2R-4 and mT2R-8) responded to denatonium and 6-n-propyl-2-thiouracil. Mice strains deficient in their ability to detect cycloheximide have amino acid substitutions in the mT2R-5 gene; these changes render the receptor significantly less responsive to cycloheximide. We also expressed mT2R-5 in insect cells and demonstrate specific tastant-dependent activation of gustducin, a G protein implicated in bitter signaling. Since a single taste receptor cell expresses a large repertoire of T2Rs, these findings provide a plausible explanation for the uniform bitter taste that is evoked by many structurally unrelated toxic compounds. Introduction Mammals can perceive and distinguish between sweet, sour, bitter, and salty tastes (Kinnamon and Cummings, 1992; Lindemann, 1996a; Stewart et al., 1997). Of these four modalities, bitter perception has a particularly important role: many naturally poisonous substances taste bitter to humans, and virtually all animal species show an aversive response to such tastants (Garcia and Hankins, 1975; Glendinning, 1994; Glendinning et al., 1999), suggesting that bitter transduction evolved as a key defense mechanism against the ingestion of harmful substances. The biology of bitter perception is very poorly understood; neither the sensory receptor cells nor the receptor molecules have been physiologically or molecularly
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defined (Lindemann, 1996b). However, several biochemical and physiological studies have suggested that bitter transduction in mammalian taste receptor cells is mediated by G proteins and G proteincoupled receptors (GPCRs) (Lindemann, 1996a; Wong et al., 1996). Because the universe of chemical compounds that evoke a bitter taste is structurally diverse, we reasoned that bitter receptors might encompass a large GPCR family with significant sequence variation. In the accompanying paper (Adler et al., 2000 [this issue of Cell]), we described the isolation of a novel family of 4080 divergent GPCRs, T2Rs, selectively expressed in subsets of taste receptor cells of the tongue and palate epithelium. T2Rs in humans and mice are genetically linked to loci associated with bitter perception (Conneally et al., 1976; Capeless et al., 1992; Reed et al., 1999), and are selectively expressed in taste receptor cells that contain gustducin, a G protein subunit implicated in bitter transduction (Wong et al., 1996; Ming et al., 1998). While the genetics, expression profile, and diversity of the T2R family support the proposal that T2Rs are taste receptors, rigorous demonstration of their role in taste transduction requires functional validation. Here we use a heterologous expression system to demonstrate that T2Rs function as receptors for bitter tastants. We analyzed mouse strains that differ in their recognition of various bitter compounds and show that mice that do not perceive low concentrations of cycloheximide contain missense mutations in the mT2R-5 gene. These amino acid changes significantly reduce the sensitivity of the mT2R-5 receptor to cycloheximide. Notably, this sensitivity shift measured in cell-based assays closely mirrors the behavio...
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