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Molecular Identification of a Taste Receptor Gene for Trehalose in Drosophila

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Science  07 Jul 2000:
Vol. 289, Issue 5476, pp. 116-119
DOI: 10.1126/science.289.5476.116

Abstract

The molecular nature of sweet taste receptors has not been fully explored. Employing a differential screening strategy, we identified a taste receptor gene, Tre1, that controls the taste sensitivity to trehalose in Drosophila melanogaster. The Tre1 gene encodes a novel protein with similarity to G protein–coupled seven-transmembrane receptors. Disruption of theTre1 gene lowered the taste sensitivity to trehalose, whereas sensitivities to other sugars were unaltered. Overexpression of the Tre1 gene restored the taste sensitivity to trehalose in the Tre1 deletion mutant. The Tre1 gene is expressed in taste sensory cells. These results provide direct evidence that Tre1 encodes a putative taste receptor for trehalose inDrosophila.

Olfactory receptors are G protein–coupled seven-transmembrane proteins encoded by a divergent multigene family in vertebrates (1) and inDrosophila (2, 3). Taste receptors are also thought to belong to the superfamily of G protein–coupled receptors (GPCRs) (4), and several candidate genes have been reported (5–8), but their function as a taste receptor has not been proved. In Drosophila, taste sensilla are present on the labellum, tarsi, and wing margins (9,10). In a typical chemosensillum on the labellum, there are four taste sensory cells each of which responds to either water, salt, or sugar. Previously, we showed that there are at least three separate receptor sites for sugars in the sugar receptor cell ofDrosophila (11, 12). The Tre gene was identified through studies on natural variants (12). Because the Tre gene controls taste sensitivity to trehalose without affecting the responses to other sugars, the gene product ofTre should function in sugar receptor cells. TheTre gene is cytologically mapped to the region between 5A10 and 5B1-3 on the X chromosome (13). A P1 clone containing a 90-kb genomic region from 5A9 to 5B1 was used as a starting material to clone the taste receptor gene (Fig. 1A).

Figure 1

(A) Tre is mapped between 5A10 and 5B1-3 on the X chromosome (13), and the P1 clone DS07361 covers this interval. (B) A restriction map of genomic region where the putative trehalose receptor gene was identified. Identified exons of the putative trehalose receptor gene are boxed. Filled and open boxes show coding and noncoding regions, respectively. The Tre1 gene is separated into eight exons. The 5′ termini of cDNA contained noncoding sequence of 247 bp. The first exon is far from the second exon for about 4 kb. Four genomic clones (#12A, #12B, #12C, and #12D) are shown. B, Bam HI; E, Eco RI; H, Hind III; N, Not I. One EP(X) line has an insertion upstream to the Tre1 coding sequence. The extent of deletion in ΔTre1 lines is shown by arrowheaded lines. (C) The topology is based on the structure of the other members of the G protein–coupled receptor family, with the NH2-terminal region extracellular. The membrane is shown by shaded box. Similarity was examined by comparing TRE1 with other GPCRs, chicken melatonin receptor, mouse neuropeptide Y receptor, human dopamine receptor, and scallop Go-coupled rhodopsin.

To identify the putative Tre gene, we performed a differential screening for genes encoded in the P1 clone that might be specifically expressed in chemosensory cells. We took advantage of thepox-neuro (poxn) gene, which is involved in the developmental decision pathway between mechanosensory and chemosensory cell fates (14). In an adult-viable allele of the poxn mutant, all external chemosensilla are either transformed into mechanosensilla or are deleted (15). In the legs of the wild-type fly, chemosensilla exist on the tarsus, but there are no chemosensilla on the femur. We performed a differential screening with cDNA probes derived from labella, tarsi, and femurs of wild-type and poxn mutant flies (16).

Southern blot analysis of the subcloned P1 DNA fragments identified one clone that hybridized to the wild-type labella and tarsi probes, but not to the other probes (17). A portion of the 8.2-kb clone displayed conserved features of the superfamily of seven-transmembrane domain receptor proteins. The full-length putativeTre1 cDNA was obtained by reverse transcriptase–dependent polymerase chain reaction (RT-PCR) and 5′ and 3′ rapid amplification of cDNA ends (RACE) (18). Sequence analysis revealed that the putative Tre1 gene contained a 1179–base pair (bp) open reading frame that encodes 392 amino acid residues preceded by an in-frame termination codon. Hydropathy analysis suggests that theTre1 cDNA sequence contains seven hydrophobic stretches that represent potential transmembrane domains (Fig. 1C). These domains constitute the regions of maximal sequence similarity to other seven-transmembrane receptors. Although several conserved regions are found between Tre1 and other GPCRs, the structures of the third and fourth cytoplasmic domains may be unique, because they are longer than the corresponding domains of typical GPCRs. TheTre1 gene has no similarity to other GPCRs recently identified by database search (8). We suggest that theTre1 gene may represent a new subclass of GPCRs.

By searching the Drosophila DNA database with the 5′-flanking genomic sequences of the putative Tre1 gene, we found that flanking genomic sequences of the P-element in strains in one of the transposon-inserted strains [EP(X) strains (19)] completely matched our genomic sequence (Fig. 1B). The EP element is inserted 113 bp upstream to the transcription initiation site in the EP(X)0496 strain. The taste sensitivity of this strain to trehalose was tested with the two-choice preference test (12) and was found to be highly sensitive (high-sensitive). We expected that imprecise excision of the P-element should disrupt the promoter region of the Tre1 gene, and this event might change the trehalose sensitivity. The EP element carriesw + as a genetic marker, and the element was jumped out by genetically supplying a transposase source (20). We tested w male flies by two-choice preference tests, using 30 mM trehalose and 2 mM sucrose as the choices. At this concentration of trehalose, nearly 98% of the parental EP(X)0496 flies preferred trehalose (Fig. 2B). Most of the w flies preferred trehalose, indicating that the precise excision of the P-element did not impair trehalose sensitivity. Flies that consumed the sucrose side were selected and individually crossed toC(1)DX attached-X females. Among about 3000 wflies, we isolated 90 lines that were confirmed as showing low sensitivity (low-sensitive) to trehalose. We determined the extent of deletion in all the 90 lines by PCR, using primers flanking the P-element insertion site. There were no amplification products in most of these lines, indicating that a deletion eliminated the primer site(s). Next, several lines were selected, and the extent of deletion was determined by Southern blotting. The results (Fig. 1B) indicated that the deletions removed the putative promoter region and the first exon. In fact, RT-PCR analyses indicated that the Tre1 mRNAs are undetectable in all these lines (Fig. 2A) (21). We determined the sequence surrounding the insertion site and confirmed that the strain that showed high sensitivity to trehalose underwent a precise excision event. The Tre1 mRNA was normal in this line (Fig. 2A, lane 7).

Figure 2

(A) Comparison of Tre1mRNA level in EP(X)0496 and ΔTre1 lines as revealed by RT-PCR. Lane 1 to 6: imprecise excision lines (trehalose low-sensitive), ΔTre1 #1 to #6. Lane 7: a precise excision line (trehalose high-sensitive). Lane 8: the original EP(X)0496. Dras was used as an internal control in RT-PCR reactions. Using Tre1-specific primers, a single 889 bp fragment was amplified. (B) Taste sensitivity curves for trehalose in EP(X)0496 and ΔTre1 lines #1 and #3. Each point represents data using 200 to 250 flies from at least five independent experiments. SEM of each point of data is within 10%. The sensitivity difference is statistically significant by ttest (P < 0.01).

We measured the taste sensitivity to trehalose of two ΔTre1 lines by the two-choice preference test with different concentrations of trehalose (Fig. 2B) (22). The sensitivity to trehalose can be defined as the PI50, the concentration of trehalose that gives a 50% preference index (PI). For Canton-S, a typical high-sensitive strain, PI50 is 10 mM. In the original EP(X)0496 flies, the PI50 value is 12 mM, whereas the value is 80 mM in the two ΔTre1 lines. Taken together, the disruption of the Tre1 gene leads to lowering the taste sensitivity to trehalose. Results of the two-choice preference test cannot discern whether trehalose sensitivity alone was altered in the ΔTre1 strains. We then examined the proboscis extension reflex by using four different sugar solutions: glucose, fructose, sucrose, and trehalose (23). The results demonstrate that the response to trehalose was specifically reduced in the ΔTre1 lines (Fig. 3). Since sensitivity to other sugars was unaffected, the sensitivity difference to trehalose should be attributed to a defect in the trehalose receptor. This conclusion is supported by the observation that the nerve responses to trehalose in the labellar chemosensilla were reduced in the ΔTre1 mutant, whereas the sucrose sensitivity was unaffected (24). This electrophysiological evidence indicated that TRE1 is directly involved in trehalose sensation.

Figure 3

The taste sensitivity to trehalose is affected in ΔTre1 lines. Proboscis extension reflex was examined using 30 males of EP(X)0496 and ΔTre1#1. Sucrose (5 mM), trehalose (15 mM), glucose (40 mM), and fructose (40 mM) were used as stimulants. Asterisk indicates significantly different by the chi-square test (P < 10–7). Different batches of flies and other ΔTre1 lines were tested, and they gave similar results.

To further confirm that the Tre1 gene is directly involved in the taste response to trehalose, we established transgenic lines carrying the hs-Tre1 cDNA gene so that Tre1 gene expression can be induced by heat shock (25). The P[hs-Tre1]#1 line showed the highest expression ofTre1 mRNA after heat shock. Heat shock was tested in the background of the ΔTre1 deletion mutant and was found to restore the trehalose sensitivity of the ΔTre1 deletion mutant (Fig. 4).

Figure 4

Induced overexpression of TRE1 restored the trehalose sensitivity in ΔTre1 flies. Two-choice preference tests were performed between 2 mM sucrose and 80 mM trehalose. ΔTre1 #1 flies carry two copies of P[hs-Tre1]#1 on the autosome. Percentages show the proportion of flies preferred the trehalose side. Flies aged 0 to 2 days after emergence were heat-shocked at 36°C for 1 hour with an interval of 24°C for 7 hours. This regime was continued for 24 hours. Flies were tested 3 hours after the last heat-shock. Error bars: SEM. Asterisk indicates significantly different by t test (P < 0.0001).

Because the Tre1 gene was isolated on the basis of its specific expression in taste sensory cells, the gene was likely to be expressed in taste cells. RT-PCR analyses on isolated labella and tarsi preparations showed that the mRNA is expressed in the labella and tarsi of original EP(X) lines but is absent in ΔTre1 andpoxn flies with no taste sensory cells. In situ hybridization experiments showed that the Tre1 mRNA is present in one of the taste sensory cells beneath a taste bristle (Fig. 5) (26). There were no signals in the labellum preparation of poxn and in the central brain. Thus, Tre1 seems to be specifically expressed in taste sensory cells.

Figure 5

Fluorescent in situ hybridizations were done on whole-mount preparations of labella. (A) A Nomarski image shows the location of taste bristles. Positive signal ofTre1 mRNA is shown as green in (B) and (C). Counter-staining of nuclei was done with propidium iodide [red (C)]. Taste sensory cells are identified by staining with monoclonal antibody 22C10 [blue (C)]. Bar, 30 μm. (B and C) Tre1mRNAs were detected in one of the sensory cells in a labellum [arrow in (B)], using an antisense probe. Colocalization of theTre1 mRNA signal in one of the taste sensory cells was observed for other cell clusters at different confocal planes. Signals along the taste bristles are nonspecific.

In summary, we have identified a putative taste receptor gene,Tre1, in Drosophila and conclude that the product of the Tre1 gene likely functions as a taste receptor for trehalose. First, disruption of the Tre1 gene lowered the trehalose sensitivity of sugar receptor cells while leaving sensitivity to other sugars intact. Second, overexpression of the Tre1transgene restored the response to trehalose. Third, theTre1 gene was specifically expressed in putative sugar receptor cells. Because the Tre1 gene identified in this study was isolated from the genomic clone where Tre is mapped, we think that the mutation(s) of the Tre1 gene are involved in the natural variation (27). If we assume that TRE1 is the sole receptor for trehalose, the null mutant ofTre1Tre1) should show no response to trehalose. The ΔTre1 flies still respond to higher concentrations of trehalose, and this response would be mediated by another unidentified receptor for trehalose, although we cannot exclude the possibility that deletion mutants are not null. In fact, we have identified two other genes in the Drosophila genome with similarity to Tre1, and we think that TRE1 belongs to a novel family of G protein–linked transmembrane receptors that may operate as taste receptors. The function of the clone gene should be investigated using expression systems, as has been successfully applied in the studies of olfactory receptors (28–31).

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: tanimura{at}rc.kyushu-u.ac.jp

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