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Colloquium Drosophila Gr5a encodes a taste receptor tuned to trehalose Sylwester Chyb*, Anupama Dahanukart, Andrew Wickens*, and John R. Carlsont: *Imperial College London, Wye Campus, Kent TN25 5AH, United Kingdom; and "Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520 Recent studies have suggested that Drosophila taste receptors are encoded by a family of G protein-coupled receptor genes compris- ing at least 56 members. One of these genes, Gr5a, has been shown by genetic analysis to be required by the fly for behavioral and sensory responses to a sugar, trehalose. Here, we show that GrSa is expressed in neurons of taste sensilla located on the labellum and legs. Expression is observed in most if not all labellar sensilla and suggests that many taste neurons express more than one receptor. We demonstrate by heterologous expression in a Dro- sophila S2 cell line that Gr5a encodes a receptor tuned to trehalose. This is the first functional expression of an invertebrate taste receptor. The majority of taste sensilla in Drosophila are located on the labellum, a gustatory organ of the proboscis, and the tarsal segments of the legs. Most sensilla house four gustatory neurons, one sensitive to sugars, one strongly sensitive to salt, one weakly sensitive to salt, and one responsive to water, as well as one mechanosensory neuron (1, 2~. Previous work has identified a large family of G protein-coupled receptor genes, the Gr genes, which have been proposed to encode gustatory receptors in Drosophila (3~. The Gr family comprises at least 56 members, many of which are expressed in subsets of taste neurons in the different taste organs (3-54. Perception of sugars is a critical taste modality in animals such as fruit flies that ingest sweet substances for nutrition. Among the sugars, one that plays a particularly important role in insects is the disaccharide trehalose ~l-O-(cx-D-glucopyranosyl-~-D- glucopyranose)], which is composed of two glucose molecules connected by an unusual 1,1' linkage. Also called mycose, this disaccharide is abundant in yeasts and fungi, which are present in fermented fruit, an important food source of Drosophila. Trehalose is especially critical to insects in that it is the principal sugar found in the hemolymph, where it is involved in regulation of osmolarity, and in many winged insects it is an easily trans- ported and accessible energy source metabolized during flight (6). Previous genetic studies have identified a locus on the X chromosome called Tre, whose alleles confer differing levels of sensitivity to trehalose (7, 8~. Recently we have shown that a member of the Gr family, GrSa, maps to this locus and is necessary for trehalose response in viva (~9~. Deletion mutants of GrSa have a greatly diminished response to trehalose when assayed by electrophysiological recordings from single taste sensilla, or by a behavioral test. This defect was rescued by reintroducing a functional copy of GrSa on a transgene, but not by introducing a mutant copy of GrSa. Consistent with our results, Ueno et al. (10) showed that polymorphisms in Gr5a correlate with trehalose responses. However, these results do not exclude the possibility that GrSa plays an indirect role in trehalose response. Here we show that GrSa is expressed in taste neurons of the labellum and the tars), supporting its identity as a taste receptor 14526-14530 1 PNAS 1 November 25, 2003 1 vol. 100 1 suppl. 2 gene. We then provide direct evidence that GrSa encodes a trehalose receptor by expressing it in Drosophila S2 cells and showing that stimulation with trehalose evokes changes in intracellular calcium (Ca2+) levels in these cells. We further show that GrSa is narrowly tuned to trehalose, showing little if any response to other disaccharides. Methods Expression Analysis. An 8.5-kb fragment upstream of GrSa was amplified from genomic DNA of Canton-S flies and inserted upstream of the GAL4-coding sequence in the pG4PN vector (C. Warr and J.R.C., unpublished data). Transgenic flies were generated by using standard procedures. Heterozygous flies carrying one copy each of GrSa-GAL4 and UAS-lacZ were stained for LacZ activity. For visualization of GFP, flies carrying two copies of GrSa-GAL4, as well as two copies of UAS- mCD8:GFP, were examined by using confocal microscopy. Heterologous Expression. A 1.2-kb fragment of full-length GrSa cDNA was amplified from head mRNA by using the Smart RACE cDNA Amplification kit (Clontech). This fragment was inserted into a unique EcoRI site in the pRmHa3 vector (11~. Drosophila S2 cells were grown at 25C in Shields and Sang M3 insect medium supplemented with 10% FCS and antibiotic/ antimycotic solution. S2 cells were cotransfected with pRmHa3-GrSa and pIZT-V5/His vector (Invitrogen) encod- ing GFP and zeocin-resistance to facilitate recognition of transfectants and antibiotic selection, at a 3:1 ratio by using liposomal formulation (CellFectin, Invitrogen) according to the manufacturer's instructions. Expression of GrSa was in- duced by adding 0.6 mM Cu2+ to the cell culture media 48 hrs before an experiment. Levels of GrSa expression in uninduced and induced cells were examined by RT-PCR to confirm the induction of Gr5a. Calcium Imaging. Transfected cells were grown in 96-well plates and loaded with membrane-permeable fura 2-acetoxymethyl ester (fura 2-AM) as described elsewhere (12~. Briefly, after being washed in Hanks' balanced salt solution (HBSS), cells were incubated for 1 h with 1-2 ,uM fura 2-AM (in 10% Pluronic F-127) at room temperature and under low light conditions. Subsequently, fura 2-AM solution was removed and cells were incubated for 1 h in HESS (50 ill) to allow endogenous esterases to cleave AM ester. Cells were stimulated by addition of 50 al of 2x tastant solution. Response kinetics were measured from cells loaded with 100 ,u M fura 2 via patch pipette and stimulated This paper results from the Arthur M. Sackier Colioquium of the National Academy of Sciences, "Chemical Communication in a Post-Genomic Worid," held January ~ 7-19, 2003, at the Arnold and Mabel Beckman Center of the National Academies of Science and Engineering in Irvine, CA. tTo whom correspondence should be addressed. E-mail: john.carlson~yale.edu. ! 2003 by The National Academy of Sciences of the USA www.pnas.org/cgi/doi/ 10.1 073/pnas.2 135339100

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c i Fig. 1. Expression of GrSa in taste neurons in the labellum and distal segments in the leg. Shown here are whole mount samples of labella or tars). Genotypes examined were as follows: Gr5a-GAL4/+; UAS-lacZ/+ (a and c) and Gr5a-GAL4/UAS-mCD8:GFP; GrSa-GAL4/UAS-mCD8:GfP(b and d9. Shown in b is a composite of a series of confocal images. Reporter gene expression was observed in #30 cells in each half of the labellum and 2 cells in each of the two to three distal-most segments of the leg. with lx test ant solution via a puffer pipette under control of PicoPump 830 (World Precision Instruments, Sarasota, FL). All chemicals were purchased from Sigma. Imaging experiments were conducted on a Nikon TE300 inverted microscope equipped with a Nikon superfluor lens ~ x 10/0.5 and x20/0.7s). Cells were stimulated with 340 and 380 nm UP light from a DeltaRAM monochromator, and resulting images were col- lected by using an IC-200 intensified charge-coupled device camera; data acquisition and analysis were performed by using an ImageMaster suite (PTI, South Brunswick, NJ). Individual responses were recorded for 60 s. F340/380 ratios were analyzed to measure Ca2+ release. A responder cell was defined as one showing a ratio of 0.34 or above. In general, >905to of responses showed a ratio of at least 0.51. Results To test the hypothesis that GrSa plays a direct role in trehalose reception, we first asked whether it is expressed in taste neurons. Because previous attempts at in situ hybridization have proved unsuccessful with the great majority of Or genes (3, 4) we generated GrSa promoter-GAL4 lines. An 8.5-kb genomic re- gion upstream of GrSa was used to supply a promoter, and GAL4 was used to drive expression of both UAS-lacZ and UAS-GEP Chyb et a/. 0.8 o ._ co o o o . . - o . . . . 60S fig. 2. Time course of trehalose response in S2-GrSa cells. (Upper) A series of images of a single fura 2-loaded S2-GrSa cell, taken at 5-s intervals. The first image is taken 5 s before the application of 100 mM trehalose. (Lower) A quantitative representation of the response of the same cell. Bar indicates stimulus period. reporters. We observed wide expression in taste neurons of the labellum as well as in four to six neurons in the tarsi of adult flies (Fig. 1~. No sexual dimorphism was observed in the expression pattern. Six independently derived lines were examined and all gave equivalent results. To examine GrSa function at the cellular level, we expressed GrSa cDNA in Drosophila S2 cells. This cell line was chosen for two reasons. First, chemosensory receptors have been notori- ously difficult to express in heterologous systems and we pre- dicted that use of a Drosophila cell line might improve the possibility of functional expression of a Drosophila receptor. Second, previous studies have documented Ca2+ release after activation of G protein-coupled receptors that couple to the endogenous Gq protein of S2 cells (13-15~. In this system, ligand binding to the receptor results in the activation of the phospho- inositide (PI) pathway: hydrolysis of PIP2 by phospholipase C into InsP3 and 4,5-diacylglycerol, and release of Ca2+ from intracellular stores. The stimulus-activated change in [Ca2+]i can be monitored with Ca2+-sensitive fluorescent ratiometric indi- cators, such as fura 2 (16~. We transiently expressed GrSa in S2 cells, loaded them with 100 ,uM fura 2, and applied 100 mM trehalose via puffer pipette (Fig. 2~. Stimulation evoked Ca2+ release: cell response developed within ~5 s of ligand application and reached a peak intensity within ~15 s of application. Upon removal of the ligand, the level of intracellular calcium gradually returned to the baseline. These data provided initial evidence that GrSa encodes a functional trehalose receptor when expressed in S2 cells. The results also suggested the possibility that GrSa- encoded receptor protein couples to the endogenous phos- phoinositide pathway. We then cotransfected S2 cells with GrSa and promiscuous G proteins: G OCR for page 14
pan me trehalose 25 AM trehalose 250 me maltose 250 EM trehalose no GrSa receptor 2.5 mM trehalose Fig. 3. Dose-dependence of trehalose response in S2-Gr5a cells. (Upper) Divided panels of 52-GrSa cells (Left and Center) or negative controls, transfected with GFPvectoralone(Right), beforeandafterapplication of either250 mMtrehalose (Leftand Right) or250mM maltose (Center). (Lower) ImagesoffieldsofS2-Gr5a cells taken on application of different concentrations of trehalose (indicated below). ,uM, and saturation was observed in the low mM range (Fig. 4a). The Hill coefficient was nH = 1.92, suggesting the possibility that GrSa functions as a homodimer. We have investigated the ligand specificity of GrSa by challenging S2-GrSa cells with other sugars. We first tested two other dissacharides, maltose (composed of two glucose units) and sucrose (one glucose unit linked to fructose), and found that even when tested at high concentrations they elicited little if any response, as measured in terms of the percentage of cells that yielded a F340/380 - 0.34 (Figs. 3 and 4~. We then systematically tested a number of common disaccharides struc- turally related to trehalose, each composed of two glucose units (Fig. 4b). These isomers varied only in the positions of their glycosidic bond (e.g., 1,1', 1,4', or 1,6') and/or the configuration (c' or ,`3) of the D-glucose subunits. None of these other disaccharides evoked significant responses (Fig. 4c). The isomers tested included isotrehalose and neotrehalose, which, like trehalose, contain 1,1' linkages; however, isotrehalose contains a I3,,B linkage, and neotrehalose contains an cz,,B linkage, whereas trehalose contains an ~x,~ linkage. D-glucose, a monosaccharide component of all of the disaccharides tested, evoked no detectable Ca2+ release even at the highest dose tested (250 mM). The simplest interpretation of these results is that GrSa recognizes moieties close to the la,l'c~ glycosidic bond. Trehalose is found in certain drought-adapted organisms such as yeasts and is thought to play a role in protecting the membrane during dehydration (19, 20~. Although trehalose has no effect on Ca2+ levels in control cells that do not express GrSa, we carried out two further experiments to control for the formal possibility that trehalose interacts with the plasma membrane and somehow activates the receptor nonspecifically. We found first that tre- halose had no effect on cells transfected with another Gr gene, Gr64f, and, second, that other molecules believed to have similar effects on membranes, glycerol and 1,2 propanediol, have no effect on GrSa-expressing cells (tested at 100 mM each, data not shown). These results are consistent with the conclusion that trehalose is a ligand for GrSa. 14528 1 www.pnas.org/cgi/doi/10. 1 073/pnas.21353391 00 D ~ Iscusslon This study provides direct evidence that GrSa, a member of a large family of G protein-coupled receptors, functions as a trehalose taste receptor. GrSa is expressed in neurons in taste sensilla of both the labellum and tars), consistent with a role as a taste receptor. When expressed in Drosophila S2 cells, it confers a response to trehalose in a dose-dependent manner. The response depends both on the expression of GrSa and on a specific stimulus, trehalose. Other disaccharides tested evoke little if any response in this system. We have provided evidence that GrSa is expressed in all, or almost all, of the ~33 sensilla present on the labellum. Because the labellum responds to a variety of sugars, and because the sensilla each contain a single sugar-sensitive neuron, the broad expression we have observed is consistent with a model in which many, if not all, of the sugar-sensitive taste neurons express more than one receptor. This model is supported by our earlier finding that mutation of GrSa affected the physiological response of the sugar cell to trehalose, but not to sucrose, as if many of the sugar-sensitive cells contain both a trehalose receptor, GrSa, and a sucrose receptor (9~. The expression pattern of GrSa is broader than that observed for previously described GAL4 lines established by using pro- moters of other Or genes (4, S). The broad pattern is consistent with our earlier physiological data (9), which indicated that Gr5a is required for trehalose response in all L- and M-type sensilla (21~. Hiroi et al. (22) also found that most sensilla on the labellum respond to trehalose. We note that the response threshold of S2-GrSa cells to trehalose is lower than in taste neurons in viva, as determined in single-unit electrophysiological recordings (9). There are several possible explanations for this difference. One is that the two experiments measure different parameters, i.e., Ca2+ levels vs. action potential frequency, and the Ca2+ level we have established as a criterion for scoring a response may be less than that required to initiate action potentials. Another possibility concerns access to tastant: in the expression system, Chyb et a/.

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a 1.25 1 C 60 1.00 a) o ~ 0.7~ a) N 0.50 o 0.2 0.00 / &=~}~ Maltose Sucrose ~ ~ Trehalose 0.025 0.25 2.5 25 250 [sugar] mM b Trehalose Isotrehalose OH HO~ H~_: OH i o 11 5~ -= 40- o Q 30- tv 2 1O ~ _ HQ ~OH b~ OH HO OH H~ H ~O~OH OH O:_ OH HC)' ~ ~ 0 ~ (D 0 a) (D ~ 0 0 ~ u' cn cn oo ~ ~ 6o u' o o o o o o o o o . 0 ~ 0 0 53 E ~ ~ C, Z ~ Z ~ Neotrehalose ,OH HO~ HO~ OH -' ~ ~O~ ~OH Maltose Cellobiose Sucrose OH H~ j OH ~\ HO~OH OH OH OH OH H~(Z~_OH OH OH Isomaltose Genbobiose Glucose OH OH H~ j H:Q: HO: OH] HO - ~ HO~_OH OH OH OH OH HO~ H(~ OH] HO~OH 1H OH Fig. 4. (a) Dos~response curves for selected disaccharides. Responses are normalized to response at 250 mM trehalose. 4 c n c 6; error bars = SEM. (b) Structures of selected disaccharides related to trehalose. With the exception of sucrose, all are composed of 2 units of glucose. The cY-configuration is highlighted in yellow, and the ,B-configuration of the C1 carbon is highlighted in red. Isotrehalose and neotrehalose do not occur naturally. (c) Specificity of trehalose response in S2-GrSa cells. Shown here are the responses of GrSa-S2 cells to the various disaccharides illustrated in b. Compounds were tested at a concentration of 250 mM. 5 c n c 9. "No GrSa" cells were stimulated with 250 mM trehalose. the cells are bathed in tastant, whereas in vivo the tastant must enter a pore in a sensillum and diffuse into the lymph surrounding a dendrite, where its final concentration may be lower than that of the test solution. A third possible explana- tion is that the density of receptor protein, or of another signaling component, may be greater in the heterologous expression system than in vivo. The simplest interpretation of our results is that GrSa func- tions as a homodimer, unlike the ~nammalian sweet receptors, which function as heterodimers (12~. Furthermore, in contrast to the TlR2/TlR3 mammalian receptor that is rather broadly Chyb et a/ furled to diverse sweet-tasting molecules such as sucrose, sac- charin, dulcin, and acesulfame-K, the GrSa receptor is tuned to trehalose and shows much less, if any, response to other sugars, such as sucrose, fructose, and glucose, which the fly encounters in its natural habitat. The relatively narrow tuning of GrSa has implications for the mechanism of taste coding. If other Drosophila taste receptors are as specific as GrSa, then an individual tastant is likely to be encoded largely by the activity of one or a small number of receptors, as opposed to the integrated activity of many recep- tors, each exhibiting a varying degree of response to a ligand. In PNAS I November25, 2003 1 vol. ~Oo 1 suppl. 2 1 14529

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the olfactory system of Drosophila, many individual odorants activate several distinct classes of receptor neurons (23, 24), each expressing distinct odor receptors (ref. 25 and J.R.C., unpub- lished results). This model of taste coding is also supported by the severe loss of trehalose response after mutation of a single receptor gene, GrSa. Analysis of further Or proteins will be required to determine whether the narrow tuning of GrSa is representative of Or receptors at large or of those that recognize 1. Dethier, V. (1976) The Hungry Fly (Harvard Univ. Press, Cambridge, MA). 2. Rodrigues, V. & Siddiqi, O. (1978) Proc. Indian Acad. Sci. Sect. B 87, 147-160. 3. Clyne, P. J., Warr, C. G. & Carlson, J. R. (2000) Science 287, 1830-1834. 4. Scott, K., Brady, R., Jr., Cravchik, A., Morozov, P., Rzhetsky, A., Zuker, C. & Axel, R. (2001) Cell 104, 661-673. 5. Dunipace, L., Meister, S., McNealy, C. & Rein, H. (2001) Curr. Biol. 11, 822-835. 6. Chapman, R. F. (1982) in The Insects: Structure and Function (Harvard Univ. Press, Cambridge, MA), 3rd Ed., p. 919. 7. Tanimura, T., Isono, K., Takamura, T. & Shimada, I. (1982) J. Comp. Physiol. 147, 433-437. 8. Tanimura, T., Isono, K. & Yamamoto, M. (1988) Genetics 119, 399-406. 9. Dahanukar, A., Foster, K, Van der Goes van Naters, W. M. & Carlson, J. R. (2001) Nat. Neurosci. 4, 1182-1186. 10. Ueno, K., Ohta, M., Morita, H., Mikuni, Y., Nakajima, S., Yamamoto, K. & Isono, K. (2001) Curr. Biol. 11,1451-1455. 11. Bunch, T. A., Grinblat, Y. & Goldstein, L. S. B. (1988) Nucleic Acids Res. 16, 1043-1061. 12. Nelson, G., Hoon, M. A., Chandrashekhar, J., Zhang, Y., Ryba, N. J. P. & Zuker, C. S. (2001) Cell 106, 381-390. 14530 1 www.pnas.org/cgi/doi/ 10.1 073/pnas.2 135339100 tastants of particular metabolic significance to the fly, such as trehalose. We thank Dr. K Moms and Mrs. M. Chyb for technical assistance and Dr. R. C. Hardie, Dr. S. Sunon, Prof. J. L. Frazier, Dr. W. M. van der Goes van Naters, and A. Ray for discussion or comments on the manuscript. This work was supported by Biotechnology and Biological Sciences Research Council (S.C.), a National Research Service Award (to A.D.), and National Institutes of Health grants and a McKnight Investigator Award (to J.R.C.~. 13. Hardie, R. C., Reuss, H., Lansdell, S. J. & Millar, N. S. (1997) Cell Calcium 21, 431-440. 14. Chyb, S., Raghu, P. & Hardie, R. C. (1999) Nature 397, 255-259. 15. Graziano, M. P., Broderick, D. J. & Tota, M. R. (1999) in Identification and Expression of G Protein-Coupled Receptors, ed. Lynch, K. L. (Wiley-Liss, New York), pp. 181-195. 16. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450. 17. Offermanns, S. & Simon, M. I. (1995) J. Biol. Chem. 270, 15175-15180. 18. Milligan, G., Marshall, F. & Rees, S. (1996) Trends Pharmacol. Sci. 17, 235-237. 19. Crowe, J. H., Crowe, L. M. & Chapman, D. (1984) Science 223, 701-703. 20. Crowe, L. M. (2002) Comp. Biochem. Physiol. 131, 505-513. 21. Ray, K, Hartenstein, V. & Rodrigues, V. (1993) Dev. Biol. 155, 26-37. 22. Hiroi, M., Marion-Poll, F. & Tanimura, T. (2002) Zool. Sci. 19, 1009-1018. 23. De Bruyne, M., Clyne, P. J. & Carlson, J. R. (1999) J. Neurosci. 19, 4520-4532. 24. De Bruyne, M., Foster, K. & Carlson, J. R. (2001) Neuron 30, 537-552. 25. Dobritsa, A. A., Van der Goes van Naters, W., Warr, C. G., Steinbrecht, R. A. & Carlson, J. R. (2003) Neuron 37, 1-20. Chyb et al.