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Umamitaste Responses Are Mediated Byα-Transducin Andα

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Umamitaste Responses Are Mediated Byα-Transducin Andα 7674 • The Journal of Neuroscience, September 1, 2004 • 24(35):7674–7680 Cellular/Molecular Umami Taste Responses Are Mediated by ␣-Transducin and ␣-Gustducin Wei He,1* Keiko Yasumatsu,3* Vijaya Varadarajan,1 Ayako Yamada,3 Janis Lem,4 Yuzo Ninomiya,3 Robert F. Margolskee,2 and Sami Damak1 1Department of Physiology and Biophysics and 2Howard Hughes Medical Institute, The Mount Sinai School of Medicine, New York, New York 10029, 3Section of Oral Neuroscience, Kyushu University, Fukuoka 812-8582, Japan, and 4Department of Ophthalmology and Program in Genetics, Tufts University School of Medicine and New England Medical Center, Boston, Massachusetts 02111 The sense of taste comprises at least five distinct qualities: sweet, bitter, sour, salty, and umami, the taste of glutamate. For bitter, sweet, and umami compounds, taste signaling is initiated by binding of tastants to G-protein-coupled receptors in specialized epithelial cells located in the taste buds, leading to the activation of signal transduction cascades. ␣-Gustducin, a taste cell-expressed G-protein ␣ subunit closely related to the ␣-transducins, is a key mediator of sweet and bitter tastes. ␣-Gustducin knock-out (KO) mice have greatly diminished, but not entirely abolished, responses to many bitter and sweet compounds. We set out to determine whether ␣-gustducin ␣ ␣ also mediates umami taste and whether rod -transducin ( t-rod ), which is also expressed in taste receptor cells, plays a role in any of the taste responses that remain in ␣-gustducin KO mice. Behavioral tests and taste nerve recordings of single and double KO mice lacking ␣ ␣ ␣ -gustducin and/or t-rod confirmed the involvement of -gustducin in bitter (quinine and denatonium) and sweet (sucrose and SC45647) taste and demonstrated the involvement of ␣-gustducin in umami [monosodium glutamate (MSG), monopotassium glutamate ␣ (MPG), and inosine monophosphate (IMP)] taste as well. We found that t-rod played no role in taste responses to the salty, bitter, and sweet compounds tested or to IMP but was involved in the umami taste of MSG and MPG. Umami detection involving ␣-gustducin and ␣ t-rod occurs in anteriorly placed taste buds, however taste cells at the back of the tongue respond to umami compounds independently of these two G-protein subunits. Key words: taste; umami; knock-out mice; gustducin; transducin; signal transduction Introduction unique because in rats, conditioned taste aversion to monoso- Bitter, sweet, salty, and sour are four widely accepted basic taste dium glutamate (MSG) generalizes to NaCl or sucrose in the qualities (for review, see Lindemann, 1996). Umami (a Japanese absence or presence of amiloride, respectively (Yamamoto et al., word meaning delicious) taste is elicited by glutamate, aspartate, 1985, 1991; Stapleton et al., 1999). some peptides, derivatives of ribonucleotides such as inosine Taste responses to bitter, sweet, and umami compounds are monophosphate (IMP) and GMP, and the metabotropic gluta- initiated by G-protein-coupled receptors (GPCRs) and trans- mate receptor agonist L-AP-4 (Sato et al., 1970; Maga, 1983; Mo- duced via G-protein signaling cascades (for review, see nastyrskaia et al., 1999; Stapleton et al., 1999). Many investigators Chaudhari and Roper, 1998; Gilbertson et al., 2000; Lindemann, consider umami as a unique fifth taste quality, based on psycho- 2001). During the past few years, several GPCRs have been iden- physical experiments in humans, conditioned taste aversion tests, tified in taste cells and implicated in taste signal transduction and genetic studies in mice, which indicate that umami is distinct (Adler et al., 2000; Chandrashekar et al., 2000; Chaudhari et al., from sweet, salty, or other taste qualities (Yoshida and Saito, 2000; Max et al., 2001; Nelson et al., 2001, 2002; Li et al., 2002). 1969; Ohara et al., 1979; Ninomiya and Funakoshi, 1989a; Bach- T1r3 plus T1r1 and a truncated type 4 metabotropic glutamate manov et al., 2000). However, others argue that umami is not receptor missing most of the N-terminal extracellular domain (taste mGluR4) have been implicated in the transduction of Received June 23, 2003; accepted July 22, 2004. umami signals in taste receptor cells (TRCs) (Chaudhari et al., This work was supported by National Institutes of Health Grants DC004766 (S.D.), DC003055 and DC003155 2000; Li et al., 2002; Nelson et al., 2002). T1r1 and T1r3 are (R.F.M.),andEY12008(J.L.)andbygrantsfromtheProgramforPromotionofBasicResearchActivitiesforInnovative coexpressed in taste buds in the anterior part of the tongue (Nel- Biosciences and International Glutamate Technical Committee to Y.N. R.F.M. is an Associate Investigator of the son et al., 2001). Taste mGluR4 is expressed in taste buds of Howard Hughes Medical Institute. *W.H. and K.Y. contributed equally to this work circumvallate and foliate papillae (Yang et al., 1999). Human CorrespondenceshouldbeaddressedtoDr.RobertF.Margolskee,DepartmentofPhysiologyandBiophysics,The embryonic kidney (HEK) 293 cells heterologously expressing Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. E-mail: [email protected]. T1r1 plus T1r3 and a promiscuous G-protein responded to glu- S. Damak’s present address: Nestle´ Research Center, Vers-chez-les-Blanc, Lausanne CH-1000, Switzerland. tamate, and this response was potentiated by IMP (Adler et al., W.He’spresentaddress:inGeniousTargetingLaboratory,25HealthSciencesDrive,StonyBrook,NY11790-3350. DOI:10.1523/JNEUROSCI.2441-04.2004 2000; Li et al., 2002; Nelson et al., 2002). At concentrations of Copyright © 2004 Society for Neuroscience 0270-6474/04/247674-07$15.00/0 MSG and L-AP-4 similar to those that produce the umami sensa- He et al. • ␣-Transducin and ␣-Gustducin Mediate Umami Taste J. Neurosci., September 1, 2004 • 24(35):7674–7680 • 7675 tion in humans, Chinese hamster ovary cells expressing taste kept on water for 7 d. The volume of liquid consumed was recorded, and mGluR4 responded by lowering cellular cAMP concentrations a ratio of tastant to total fluid consumed was determined. The data were (Chaudhari et al., 2000). The G-proteins that couple these poten- analyzed using the general linear model repeated measures of the statis- tial umami receptors to intracellular signaling pathways have not tics package SPSS with concentrations as dependant variables and geno- been identified. type as a fixed factor. When a statistically significant difference among the means was found, the Tukey’s test was used to determine which Among the G-protein ␣ subunits known to be selectively ex- ␣ means differed. For additional details of the two-bottle preference tests, pressed in TRCs is -gustducin (McLaughlin et al., 1992). see the report by Wong et al. (1996). ␣ -Gustducin shares 80% identity with cone and rod Nerve recordings. Recordings from the chorda tympani (CT) and the ␣-transducins (McLaughlin et al., 1992). ␣-Gustducin knock- glossopharyngeal nerves (NGs) were performed as described previously out (KO) mice showed strongly reduced, but not completely (Kawai et al., 2000). Tastants were applied to the tongue for 30 sec (CT) abolished, behavioral and nerve responses to several bitter and or 60 sec (NG) at a regular flow rate. Integrated whole-nerve response sweet compounds (Wong et al., 1996), indicating that magnitudes (time constant, 1 sec) were measured 5, 10, 15, 20, and 25 sec ␣-gustducin plays a key role in the transduction of these tastants. (for the CT) and 5, 10, 20, 30, and 40 sec (for the NG) after stimulus onset ␣ ␣ and averaged. These averages were normalized to the responses to NH4Cl Rod -transducin ( t-rod) also is expressed in TRCs, albeit at much lower levels than is ␣-gustducin (Ruiz-Avila et al., 1995; and analyzed with the general linear model multiple measures of SPSS with concentrations as within-subjects variables and genotypes as Yang et al., 1999). In vitro, ␣ , like ␣-gustducin, binds G␥13, t-rod between-subjects factors. can be activated by bitter-responsive taste receptors, and can ac- tivate taste-expressed phosphodiesterase isoforms (Ruiz-Avila et ␣ ␣ al., 1995). -Gustducin null mice expressing t-rod as a transgene Results driven by the ␣-gustducin promoter partially recovered re- In comparison with WT mice, ␣-gustducin KO mice have dimin- sponses to sweet and bitter compounds, indicating that the func- ished, but not abolished, responses to bitter and sweet com- tions of these two G-protein subunits in taste may overlap (He et pounds (Wong et al., 1996). Furthermore, expression of a ␣ ␣ ␣ al., 2002). Despite the presence of t-rod in taste cells and its dominant-negative form of -gustducin from the -gustducin biochemical similarity to ␣-gustducin, it has never been shown promoter in ␣-gustducin KO mice further reduces their re- ␣ directly that endogenous t-rod plays a role in taste. To determine sponses to bitter and sweet compounds, indicating that other ␣ the role of t-rod in vivo in taste responses, we performed behav- taste-expressed G-proteins couple with bitter and sweet- ioral and electrophysiological tests with KO mice lacking responsive taste receptors (Ruiz-Avila et al., 2001). ␣ ␣ ␣ -gustducin and/or t-rod. We determined that t-rod is not in- ␣ ␣ volved in responses to bitter, salty, or sweet compounds but is -Gustducin, but not t-rod , mediates bitter and sweet taste involved in responses to two umami compounds [MSG and To determine whether the residual responses of the ␣-gustducin ␣ monopotassium glutamate (MPG)]. We determined also that KO mice are mediated by t-rod, we performed two-bottle pref- ␣-gustducin is a key mediator of umami taste responses. erence tests with two bitter compounds, denatonium benzoate and quinine sulfate, and two sweet compounds, sucrose and the Materials and Methods artificial sweetener SC45647 (Fig. 1). We compared responses of KO mice. The design and production of ␣-gustducin and ␣ KO mice ␣ Ϫ Ϫ ␣ Ϫ Ϫ t-rod -gustducin KO (gus / ), t-rod KO (trans / ), double KO have been described (Wong et al., 1996; Calvert et al., 2000). Double (gusϪ/Ϫ transϪ/Ϫ), and doubly heterozygous littermate WT Ϫ Ϫ Ϫ Ϫ ␣ Ϫ Ϫ ␣ KOs (gus / trans / ), t-rod KOs (trans / ), -gustducin KOs ϩ Ϫ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ control (gus / trans / ) mice.
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