Gustatory receptor 5a: Biological Overview | Developmental Biology | Effects of Mutation | References

Gene name - Gustatory receptor 5a

Synonyms - Trehalose-sensitivity

Cytological map position - 5A11-B1

Function - transmembrane receptor

Keywords - taste receptors, G-protein coupled receptor

Symbol - Gr5a

FlyBase ID: FBgn0003747

Genetic map position - 1-13.6

Classification - G-protein coupled receptor

Cellular location - transmembrane



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

Recent literature
Miyazaki, T., Lin, T. Y., Ito, K., Lee, C. H. and Stopfer, M. (2015). A gustatory second-order neuron that connects sucrose-sensitive primary neurons and a distinct region of the gnathal ganglion in the Drosophila brain. J Neurogenet: 1-26. PubMed ID: 26004543
Summary:
This study identified and characterized a bilateral pair of gustatory second-order neurons in Drosophila. GRASP (GFP reconstitution across synaptic partners) was combined with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate gustatory second-order neurons (G2Ns) and the axonal terminals of Gr5a-expressing gustatory sensory neurons GSNs, which are known to respond to sucrose. Results of this analysis revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, fluorescence-tagged acetylcholine receptor subunit (Dalpha7) and active-zone marker (Brp) were expressed in the G2Ns. G2N input sites were found to overlay GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons' arborizations. The identified G2Ns were found to receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the gnathal ganglia (GNG), and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). These findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.

Taste receptors are likely to belong to the superfamily of G protein-coupled receptors (GPCRs). In Drosophila, taste sensilla are present on the labellum, tarsi, and wing margins. In a typical chemosensillum on the labellum, there are four taste sensory cells, each of which responds to either water, salt, or sugar. The Trehalose-sensitivity (Tre) gene was identified through studies on natural variants (Tanimura, 1982). The Tre gene has been cytologically mapped to the region between 5A10 and 5B1-3 on the X chromosome (Tanimura, 1988). Because the Tre gene controls taste sensitivity to trehalose without affecting the responses to other sugars, the gene product of Tre should function in sugar receptor cells. Disruption of the Tre gene lowers the taste sensitivity to trehalose, whereas sensitivities to other sugars are unaltered. Overexpression of the Tre gene restores the taste sensitivity to trehalose in the Tre deletion mutant. The Tre gene has been shown to be expressed in taste sensory cells. These results provide direct evidence that Tre encodes a putative taste receptor for trehalose in Drosophila (Ishimoto, 2000). An independent study has identified a family of taste receptors in Drosophila that is unrelated to the Trehalose-sensitivity gene described here (Clyne, 2000).

This review is divided into two halves. The first describes the identification of CG3171 as Tre by Ishimoto (2000). This identification has been called into question by Ueno (2001). Ueno's identification of Tre as the gene adjacent to CG3171, the gustatory receptor Gr5a, is presented in the second half of this essay (see below).

To identify the putative Tre gene, a differential screen was performed for genes cloned into P1 vectors that might be specifically expressed in chemosensory cells. Advantage was taken of the pox-neuro (poxn) gene, which is involved in the developmental decision pathway between mechanosensory and chemosensory cell fates. In an adult-viable allele of the poxn mutant, all external chemosensilla are either transformed into mechanosensilla or are deleted. In the legs of the wild-type fly, chemosensilla exist on the tarsus, but there are no chemosensilla on the femur. A differential screening was carried out with cDNA probes derived from labella, tarsi, and femurs of wild-type and poxn mutant flies. The purpose of this screen was to distinguish genes expressed in wild-type but not expressed in poxn mutants (Ishimoto, 2000).

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. A portion of the 8.2-kb clone displayed conserved features of the superfamily of seven-transmembrane domain receptor proteins. The full-length putative Tre1 cDNA was obtained by reverse transcriptase-dependent polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (Ishimoto, 2000).

By searching the Drosophila DNA database with the 5'-flanking genomic sequences of the putative Tre gene, flanking genomic sequences of the P-element were found in one previously isolated transposon-inserted strain [EP(X)] that completely matched the genomic sequence of Tre. The EP element is inserted 113 bp upstream of 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 and the strain was found to highly sensitive, suggesting that the P-element insertion leaves the gene function intact (Ishimoto, 2000).

One way to isolate a mutation is to screen for imprecise excision of a P-element inserted near the gene that one wants to disrupt. It was expected that imprecise excision of the P-element should disrupt the promoter region of the Tre gene, and this event might change sensitivity to trehalose. The EP element carries w+ as a genetic marker, and the element was jumped out by genetically supplying a transposase source. w male flies were tested by two-choice preference tests, using as choices 30 mM trehalose and 2 mM sucrose. At this concentration of trehalose, nearly 98% of the parental EP(X)0496 flies preferred trehalose. Most of the w flies preferred trehalose, indicating that the precise excision of the P-element does not impair trehalose sensitivity. Flies that consumed the sucrose side were selected and individually crossed to C(1)DX attached-X females. From about 3000 w flies, 90 lines were isolated that were confirmed as showing low sensitivity to trehalose. The extent of deletion was determined 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) (Ishimoto, 2000).

Next, several lines were selected, and the extent of deletion was determined by Southern blotting. The results indicate that the deletions removed the putative promoter region and the first exon. In fact, RT-PCR analyses indicate that the Tre mRNAs are undetectable in all these lines. The sequence surrounding the insertion site was determined and it was confirmed that the strain that showed high sensitivity to trehalose had undergone a precise excision event. Tre mRNA is normal in this line (Ishimoto, 2000).

The taste sensitivity to trehalose of two DeltaTre lines was tested by the two-choice preference test with different concentrations of trehalose. 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 DeltaTre lines. Taken together, the disruption of the Tre gene leads to a lowering of the taste sensitivity to trehalose. Results of the two-choice preference test cannot discern whether trehalose sensitivity alone is altered in the DeltaTre strains. The proboscis extension reflex was examined by using four different sugar solutions: glucose, fructose, sucrose, and trehalose. The results demonstrate that the response to trehalose is specifically reduced in the DeltaTre lines. Since sensitivity to other sugars is 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 are reduced in the DeltaTre mutant, whereas the sucrose sensitivity is unaffected. This electrophysiological evidence indicates that Tre is directly involved in trehalose sensation (Ishimoto, 2000).

To further confirm that the Tre gene is directly involved in the taste response to trehalose, transgenic lines were established carrying the hs-Tre cDNA gene so that Tre gene expression could be induced by heat shock. The P[hs-Tre]#1 line shows the highest expression of Tre mRNA after heat shock. Heat shock was tested in the background of the DeltaTre deletion mutant and was found to restore the trehalose sensitivity of the DeltaTre deletion mutant (Ishimoto, 2000).

In summary, a putative taste receptor gene, Tre, has been identified in Drosophila and the product of the Tre gene likely functions as a taste receptor for trehalose. Three findings are noted: (1) disruption of the Tre gene lowers the trehalose sensitivity of sugar receptor cells while leaving sensitivity to other sugars intact; (2) overexpression of the Tre transgene restores the response to trehalose; (3) the Tre gene is specifically expressed in putative sugar receptor cells. Because the Tre gene identified in this study was isolated from the genomic clone where Tre was initially mapped mapped, it is thought that the mutation(s) of the Tre gene is involved in the natural variation. If it is assumed that Tre1 is the sole receptor for trehalose, the null mutant of Tre (DeltaTre) should show no response to trehalose. The DeltaTre flies still respond to higher concentrations of trehalose, and this response would be mediated by another unidentified receptor for trehalose, although the possibility cannot be excluded that deletion mutants are not null. In fact, two other genes in the Drosophila genome have been identified with similarity to Tre; it is thought that TRE1 belongs to a novel family of G protein-linked transmembrane receptors that may operate as taste receptors. The function of the cloned gene should be investigated using expression systems, as has been successfully applied in the studies of olfactory receptors (Ishimoto, 2000).

The identification of CG3171 as Tre by Ishimoto (2000) has been called into question by Ueno (2001). Ueno's identification of Tre as the gene adjacent to CG3171, the gustatory receptor Gr5a, is presented below. The argument is rather compelling, but the two sets of data are still in conflict because of Ishimoto's convincing overexpression data. Nevetheless, a confirmation of Ueno's result has been provided by Dahanukar (2001), who has also identified Gr5a as Tre. Dahanukar's results are given in the Effects of Mutation section.

Drosophila taste gene Tre is located on the distal X chromosome and controls gustatory sensitivity to a subset of sugars. Two adjacent, seven-transmembrane domain genes near the Tre locus are candidate genes for Tre. One (CG3171) encodes a rhodopsin family G protein receptor, and the other (Gr5a) is a member of a chemosensory gene family encoding a putative gustatory receptor. Molecular analyses of mutations in Tre were carried out in order to elucidate their involvement in the gustatory phenotype. Tre mutations induced by P element-mediated genomic deletions disrupt Gr5a gene organization and the expression of Gr5a mRNA, while disruption of the CG3171 gene or its expression is not always associated with mutations in Tre. In flies with the spontaneous mutation Tre01, both CG3171 and Gr5a mRNAs are transcribed. Coding sequences of these two candidate genes were compared among various strains. A total of three polymorphic sites leading to amino acid changes in CG3171 were not correlated with the gustatory phenotype. Among four nonsynonymous sites in Gr5a, a single nucleotide polymorphism leading to an Ala218Thr substitution in the predicted second intracellular loop cosegregated with Tre01. Taken together, the mutation analyses support that Gr5a is allelic to Tre (Ueno, 2001).

Flies carrying a spontaneous mutation Tre01 show a decreased gustatory sensitivity to trehalose. Two seven-transmembrane receptor genes Gr5a (Scott, 2001 and Dunipace, 2001) and CG3171 (Ishimoto, 2000) have been identified in the region where the Tre01 is mapped. A single P element insert [EP(X)496] (GenBank accession number AQ025347) that maps directly between the two genes was used to generate Tre mutations by activating imprecise P element excisions. A total of 22 independent Tre mutations were recovered. The gustatory phenotype has been assessed by a feeding preference test. Males of a wild-type Tre+ strain, w cx Tre+, produced the mean proportion of flies choosing the trehalose solution, or preference index (PI), of 0.90, while w cv Tre01 males carrying a spontaneous mutation Tre01 gave a low PI value of 0.30 (p < 0.01). EP(X)496 was wild-type for Tre, since the strain gave a PI value of 0.95. Males carrying the P excised Tre mutations TreDeltaEP3, TreDeltaEP5, or TreDeltaEP19 gave significantly low PIs of 0.06 or less (p < 0.01). Complementation tests between the P mutations and Tre01 confirm that the P mutations are allelic to Tre, but these P mutations of Tre apparently show a more severe phenotype than the spontaneous mutation Tre01 (p < 0.01) (Ueno, 2001).

Induced Tre mutations are genomic deletions uncovering CG3171 and/or Gr5a. The two Tre candidates CG3171 and Gr5a are adjacent genes on the genome facing the 5' end (GenBank accession number AE003435). The insertion of EP(X)496 is located between the two genes and is less than 0.1 kbp upstream of the transcription start site (GenBank accession number AB042625) of CG3171 and 0.7 kbp upstream of the putative start codon of Gr5a. TreDeltaEP3 (GenBank accession number AB066610) has a 2.1 kbp genomic deletion uncovering 0.7 kbp toward Gr5a and 1.4 kbp toward CG3171. In TreDeltaEP5 (GenBank accession number AB066611), a 2.5 kbp deletion was identified that spans 0.9 and 1.6 kbp in the directions of Gr5a and CG3171, respectively. The gene structure of CG3171 in TreDeltaEP3 and TreDeltaEP5 is disrupted by the absence of the promoter, exon 1, and part of intron 1. Gr5a gene is also disrupted in TreDeltaEP5 since it uncovers the promoter, the 5' leader, and 133 bp downstream of the start codon. Deletion in TreDeltaEP3 leaves 44 bp intact in the 5' leader, but at least 58 bp are truncated in addition to the deletion of the promoter. Therefore, TreDeltaEP3 and TreDeltaEP5 are expected to be double mutations of both CG3171 and Gr5a (Ueno, 2001).

TreDeltaEP19 (GenBank accession number AB066612) is distinct from TreDeltaEP3 or TreDeltaEP5 since the 3' end of the P element has been precisely excised out, leaving the CG3171 gene structure intact. TreDeltaEP19 uncovers a 1.0 kbp sequence in the direction of Gr5a that includes the promoter, the 5' leader, and a 253 bp sequence of the entire exon 1, intron 1, and part of exon 2. Therefore, only the Gr5a gene structure is specifically and most severely disrupted in TreDeltaEP19. Taking the genomic analyses together, disruption of Gr5a but not CG3171 is associated with Tre mutations (Ueno, 2001).

The transcripts of CG3171 and Gr5a were then investigated in the P excised mutants. RT-PCR with total RNAs isolated from the head amplified a CG3171 mRNA sequence in Tre+ and Tre01 flies. No amplification was observed with TreDeltaEP3 or TreDeltaEP5 templates. With TreDeltaEP1, TreDeltaEP13, and TreDeltaEP18 templates, the RT-PCR also failed to amplify the fragment. However, there was another class of mutations in which the expression of CG3171 mRNA was observed. TreDeltaEP7, TreDeltaEP11, TreDeltaEP12, TreDeltaEP14, and TreDeltaEP19 belong to this class. Among these, TreDeltaEP19 was predicted to express CG3171 from the intact genomic structure. In the Northern blot analysis, the antisense RNA probe of CG3171 labeled a 1.6 kbp mRNA from wild-type heads. The 1.6 kbp band was not detectable in TreDeltaEP3 or TreDeltaEP5, but was present in TreDeltaEP19, as in the RT-PCR analysis. CG3171 mRNA was further analyzed in various wild-type tissues (Ueno, 2001).

CG3171 mRNA is normally expressed in the labella, the head, and the appendages of a mutant poxn in which chemosensory neurons are transformed to mechanosensory neurons. The low tissue specificity of CG3171 is inconsistent with the claim of Ishimoto (2000) that CG3171 mRNA was identified from the differential screen of cDNA libraries between wild-type and poxn mutant tissues. A CG3171 cDNA clone LD12308 (GenBank accession number AA438512) was isolated from an embryonic library. CG3171 mRNA is expressed throughout embryonic and adult stages. A different line of evidence supporting CG3171 as a developmental gene was provided by Toba (1999) who showed that developmental phenotypes are induced in various tissues of the transformants carrying a misexpression construct inserted adjacent to CG3171 locus when the expression is activated by Gal4 drivers (Ueno, 2001).

Gr5a mRNA was examined in wild-type and mutant flies. RT-PCR was successful with poly(A)+ RNA prepared from 300 heads and 100 labella. An 856 bp Gr5a mRNA fragment was not identified in the deletions TreDeltaEP3, TreDeltaEP5, and TreDeltaEP19, while Gr5a mRNA is transcribed in wild-type, EP(X)496, and Tre01 heads. It is therefore suggested that intact mRNAs are not transcribed in those mutants. Gr5a mRNAs from wild-type and poxn labella were compared by the RT-PCR. Gr5a mRNA was identified in the wild-type labella, as observed in the heads. In the poxn labella, however, it was either absent or present in severely decreased amounts, suggesting that Gr5a is predominantly expressed in the gustatory sensory neurons in the labella. The expression analyses support that Gr5a is expressed in the gustatory neurons and that the mutation of Gr5a is associated with the Tre phenotype (Ueno, 2001).

Both CG3171 and Gr5a mRNAs were identified in the flies carrying the spontaneous mutation Tre01. The nucleotide polymorphisms leading to amino acid changes were investigated. A 1.5 kbp genomic sequence downstream of the start codon of CG3171 was analyzed in w cx Tre+, w cv Tre01, and four isofemale wild strains. The gustatory phenotype was also investigated for the wild strains. One strain (HG84) was Tre+, and the other three strains were Tre01. Sequence analysis has revealed a total of 12 single-nucleotide polymorphisms (SNPs) and an 18 bp insertion/deletion in exons. In addition, a total of 18 SNPs and two oligonucleotide insertions/deletions were also identified in the introns (GenBank accession numbers AB066613). The insertion of the 18 bp oligonucleotide ATGGATATGGATATGGGA, leading to an insertion of six amino acids in the N-terminal region, was identified in w cv Tre01 and two wild strains, HG84(Tre+) and Singapore (Tre01). In these three strains, a SNP in exon 2 leading to a Phe12Ile substitution was also identified. A second nonsynonymous SNP in exon 7 leading to a Leu348Met substitution was identified in all strains, except in w cx Tre+. None of the three sites, however, was linked to the gustatory dimorphism (Ueno, 2001).

Similarly, Gr5a polymorphisms were also analyzed for the 1.7 kbp genomic region in the following six strains: w cx Tre+, Canton-S (Tre+), HG84 (Tre+), w cv Tre01, Oregon-R (Tre01), and Singapore (Tre01). There were a total of 25 SNPs within the exons of the Gr5a gene. Nineteen SNPs and two single-nucleotide insertions/deletions were also found in the introns (GenBank accessions numbers AB066619-24). Among the exon polymorphisms, four nonsynonymous SNPs were identifed. The SNPs Met23Ile and Leu216His were identified only in Oregon-R (Tre01) and w cv Tre01, respectively. Val19Ile was identified in all three Tre01 strains but also in HG84 (Tre+). The only SNP that cosegregated with the Tre phenotype was Ala218Thr. Additional experiments have shown that Ala218Thr is significantly correlated with the gustatory Tre phenotype. The Ala218 is located in the predicted second intracellular loop domain of GR5a. Since the second or the third loop is known to be critically important in the activation of G protein both by rhodopsin and by mGluR1, GR5a may activate G protein through a similar mechanism, and the Ala218 may be involved in the activation process. Although functional characterization of Gr5a has yet to be performed, it is proposed that Gr5a encodes a gustatory sugar receptor controlled by the locus Tre (Ueno, 2001).

By Clustal W analysis, Gr5a has been shown to be most closely related phylogenetically to Drosophila candidate gustatory receptor gene Gr61a and a gene cluster of Gr64a-f on the third chromosome. Since Tre affects the taste response to a limited subset of sugars, some of these receptors may also be involved in the sweet taste response to different subsets of sugars. Physiological studies show that the input from sugar-sensitive neurons in the labellar, tarsal, and other gustatory organs controls the proboscis extension reflex and the feeding behavior. GRs have been shown to be expressed in gustatory neurons in distinct subsets of gustatory organs. Future studies on the localization and the projection of gustatory neurons expressing GR5a and the related GR members may provide clues to understanding the neuronal mechanism underlying feeding behavior in flies (Ueno, 2001).

Taste perception and coding in Drosophila

Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. The expression of eight Gr genes in the labial palps, the fly's main taste organ, has been characterized in detail. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities -- the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. It is concluded that discrimination between two taste modalities -- sweet and bitter -- requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons (Thorne, 2004).

The labellum, considered to be the main taste organ in Drosophila, has approximately 62 chemosensory bristles (sensilla) that are arranged in a stereotyped pattern. These sensilla are morphologically identified as short (S), intermediate (I), and long (L). S and L bristles house dendrites of four chemosensory neurons, whereas I bristles are associated with two chemosensory neurons. To determine expression of Gr genes in these chemosensory neurons, the Gal4/UAS system has been used. This indirect method of expression analysis has proven far superior to RNA in situ hybridization due to low levels of Gr transcripts per cell and the wide distribution of taste neurons in tissues not amenable to sectioning procedures. The Gal4/UAS analyses revealed that a given Gr gene is expressed in a small number of chemosensory neurons per labial palp and, in each case, in only one neuron per chemosensory bristle. Hiroi (2002) demonstrated an association of specific Gr genes with certain bristles of the labellum. The majority of receptors examined are expressed in one of the four neurons of S type sensilla. For example, several Gr genes are strongly expressed in a single neuron associated with three S type sensilla (S1, S3, and S6) (Thorne, 2004).

Several issues with broad implications for taste coding remain to be elucidated. For example, it is still not known whether some Gr genes are coexpressed in the same neurons and, if so, to what extent. Similarly, it is not known what kind of taste properties are mediated by GRNs expressing these receptors. Finally, experiments to visualize axonal targets in the CNS of neurons expressing individual Gr genes have not been performed in any detail. To further advance understanding of Drosophila taste perception, these questions were addressed: the number of neurons expressing novel and previously characterized Gr genes was addressed, their extent of coexpression was investigated, the projection patterns of GRNs expressing these genes was visualized, and taste perception of flies lacking specific sets of GRNs was determined (Thorne, 2004).

Gal4 drivers (p[Gr]-Gal4) for eight Gr genes, Gr5a, Gr22b, Gr22e, Gr22f, Gr28be, Gr32a, Gr59b, and Gr66a, were combined with a UAS-nucGFP reporter gene encoding a green fluorescent protein tagged with a nuclear localization signal and images of optical sections through the entire labellum were collected by using confocal microscopy after anti-GFP antibody staining. By using the map generated by Hiroi (2002) as a guide, detailed analysis of confocal stacks allowed the organization and number of neurons expressing each of these genes to be more accurately determined. The expression patterns fell into two broad groups: Gr5a (representing the first group) was expressed in a large number of neurons throughout the entire labial palp, whereas the other Gr genes had restricted expression to relatively few neurons (Thorne, 2004).

Of the second group, Gr66a was expressed in the largest number (n = 22 ± 1)of cells per palp. Significantly, only a single neuron per S and I type sensillum stained positive for this driver. The neurons associated with S type bristles, which are located more medially, appeared larger in size compared to more laterally located neurons of I type sensilla. Gr22b, Gr22e, Gr22f, Gr28be, Gr32a, and Gr59b were expressed in fewer neurons than Gr66a. Expression of these receptors appears more restricted to larger neurons associated mostly with S type bristles. These expression studies provided the groundwork necessary to determine whether two or more Gr genes are actually coexpressed in the same neuron associated with an S type bristle (Thorne, 2004).

Ideally, coexpression of Gr genes may be addressed by labeling individual Gr gene-specific probes with different markers. However, expression levels of these genes are too low for reliable detection of transcripts by RNA in situ hybridization. Attempts were made to use the Gal4/UAS system along with a second reporter system, the tetracycline transactivator/tet-O reporter system. The sensitivity of this system, however, was too low to obtain reliable cell staining in taste neurons. Therefore, the issue of coexpression was addressed by quantification of labeled cells using the Gal4/UAS system, an approach that seemed feasible given the relatively low number of cells in which each receptor is expressed. Transgenic fly lines were made expressing UAS-nucGFP under the control of two different Gal4 drivers and then the number of labeled neurons was counted and compared to that of flies containing each driver alone. Surprisingly, in all cases where such double-driver experiments were carried out, the number of labeled cells expressing two drivers was close or equal to the number of labeled cells of flies containing the single driver with the higher cell count. For example, in flies that express either the p[Gr66a]-Gal4 or p[Gr22e]-Gal4 driver, an average of 22 and 14 neurons/labial palp are labeled, respectively. In flies that express both drivers, again approximately 22 neurons are detected per palp, which indicates that most if not all cells that express Gr22e also express Gr66a (Thorne, 2004).

A crucial determinant for discerning chemical cues present in the environment is embedded in the peripheral expression pattern of cell surface receptors in sensory epithelia. In the olfactory systems of Drosophila and mice, each olfactory receptor neuron expresses only one of 60 or one of approximately 1000 Or genes, respectively, enabling these animals to discriminate between hundreds or thousands of different odors. In contrast, taste cells of the tongue allow mammals to distinguish only a few taste qualities: bitter, sweet, umami, salty, and acidic taste. Lack of discrimination between the hundreds of diverse chemical compounds -- all perceived as bitter -- is thought to be caused by coexpression of the approximately 40 T2R receptors in a single set of taste cells (Chandrashekar, 2000; Zhang, 2003). Therefore, activation of the bitter taste cells by any one of the T2Rs is likely to generate a single activation pattern in taste centers of the brain, leading to a similar, repulsive behavioral output. Associating primary taste centers in the mammalian brain with specific taste modalities has, as of yet, proved challenging (Thorne, 2004).

Insect taste is still rather poorly understood, especially at the molecular level. Drosophila, which exhibits remarkably similar taste preferences with humans, is the only insect for which candidate receptors have been characterized experimentally. The investigations presented here provide significant new insights into insect taste perception (Thorne, 2004).

Initial expression studies suggested that the fly gustatory receptors are not simply coexpressed in three sets of cells dedicated to bitter, sweet, and umami taste like the T2Rs, T1R2/T1R3, and T1R1/T1R3 receptors of mammals. Instead,these experiments suggested that they either are expressed according to the one receptor (the 'one neuron' hypothesis well established for insect and mammalian olfactory systems) or they are expressed in partially overlapping sets of neurons. The current analysis supports the latter of these possibilities. Most labellar Gr genes (seven out of eight) are expressed in a single neuron of mostly S and some I type bristles. Most interestingly, coexpression studies provide evidence that individual neurons express anywhere from one to six receptors. In this way, S bristle-associated neurons are defined by unique receptor gene codes, thereby outfitting the labellum with an array of sensory assemblies that may exhibit distinct, albeit overlapping, ligand specificities (Thorne, 2004).

The functional implications of distinct neuronal receptor codes on taste perception are currently unclear and will require analysis of mutations of individual Gr genes. However, a general role for these neurons in feeding inhibition ('avoidance neurons') can be inferred from experiments presented in this study and supported through analogy with the mammalian taste system/receptors. (1) Avoidance neurons express the majority of analyzed Gr genes -- and by extension -- the majority of the genes in the entire Gr gene family. In mammals, bitter taste receptors far outnumber the sweet taste receptors (40:3). (2) Avoidance neurons associated with S type bristles do not express the receptors for the sugar trehalose encoded by the Gr5a gene. In fact, avoidance neurons associated with S type bristles have a distinct appearance compared to neurons expressing the Gr5a gene. In mammals, the sweet/umami taste receptors and the bitter taste receptors are expressed in distinct group of cells (Chandrashekar, 2000; Zhang, 2003). (3) Inactivation of avoidance neurons has no effect on sucrose or trehalose sensitivity in flies but significantly reduces their sensitivity to caffeine. (4) Avoidance neurons and Gr5a-expressing neurons have distinct targets in the subesophageal ganglion (SOG), a feature consistent with the detection of different taste qualities by these neurons (Thorne, 2004).

If the avoidance neurons have a general function in the detection of toxic or otherwise undesirable chemicals, what is the rationale for a complex and distinct Gr gene code among different groups of such neurons? It is proposed that the receptor code allows a fly to discriminate among different chemicals, which are in general avoided but might have distinct consequences on their health if ingested. According to such a proposal, a fly encountering a food source rich in nutrients (sugars) but contaminated with toxic chemicals may choose between feeding and avoidance, depending on the impact the particular toxic compound may have on its health. There is indirect evidence from feeding studies in Maduca sexta larvae that discrimination between the bitter substrates caffeine and aristolochic acid does occur in insects, even though actual taste preference, adaptation, or both may contribute to this phenomenon (Glendinning, 2001). Thus, discrimination among toxic/bitter-tasting compounds might be possible in insects including Drosophila (Thorne, 2004).

It was somewhat surprising that the sensitivity to other compounds known to be avoided by insects -- denatonium benzoate, quinine hydrochloride, and berberine -- was not affected in animals lacking Gr66a-expressing neurons. This may simply be explained by the presence of additional neurons expressing receptors that recognize these particular substrates. Alternatively, one or a few neurons coexpressing Gr66a along with a receptor for one (of these) ligand(s) might not have been completely inactivated by TNT. Finally, studies in rodents indicate that caffeine may directly affect neurons in the brain, circumventing activation of taste cells altogether. This is not likely to be the case in the current experiments, because none of the Gr genes examined is expressed in the CNS (Thorne, 2004).

Relatively few studies have investigated bitter taste sensitivity in insects, particularly Drosophila. Electrophysiological studies have identified bristles in the legs, but not the labellum of Drosophila, that respond to bitter-tasting chemicals (Meunier, 2003). However, S type sensilla are notoriously difficult to record from, because their bristles are extremely difficult to access for this type of experiment (Thorne, 2004).

Gr5a-expressing neurons represent more than half of chemosensory cells in the labellum and appear to be associated with all sensilla types, including the taste pegs. In fact, association of Gr5a with taste pegs provides the best evidence yet that these sensilla have a specific chemosensory function in the detection of trehalose. Significantly, Gr5a-expressing neurons define a largely distinct set of neurons from the avoidance neurons. This observation is consistent with the results from behavioral investigations of flies lacking the function of specific sets of neurons. Specifically, inactivation of Gr5a-expressing neurons leads to a reduction in trehalose sensitivity, but the sensitivity to any bitter substrate tested was unaffected. These flies did not exhibit reduced sucrose sensitivity, another nutrient-relevant sugar for Drosophila. This result is somewhat unexpected, since electrophysiological investigations have led to the proposal that a single neuron in L, I, and S bristles is responsive to several sugars including trehalose and sucrose (the 'sugar' neuron) (Dahanukar, 2001; Hiroi, 2002; Rodrigues, 1981; Dethier, 1976). According to these studies, sugar neurons may express a single, broadly tuned sugar receptor, or more likely, they may coexpress several distinct sugar receptors, each of which recognizes a specific sugar (i.e., sucrose, trehalose, fructose, etc.). This latter possibility is favored from genetic studies, which have shown that mutation in the Gr5a gene reduces the sensitivity of flies to trehalose, but not to sucrose. However, the proposition of a single sugar neuron per bristle is also not consistent with expression studies, which show that two to three neurons within a bristle can express Gr5a. The possibility that the p[Gr5a]-Gal4 drivers do not represent endogenous Gr5a expression cannot be excluded, but this is unlikely to be the case for two reasons: (1) several lines with p[Gr5a]-Gal4 show the same expression, and (2) the p[Gr5a_C]-Gal4 driver containing a much larger promoter fragment produces a similar expression profile, with many clusters of Gr5a-expressing neurons associated with the same bristle (Thorne, 2004).

In order to realign the electrophysiological data with expression analysis, another explanation is proposed: the 'sugar neuron' identified in electrophysiological studies expresses many (possibly all) distinct sugar receptors, including GR5a. However, one or two additional neurons per bristle express only a fraction, or possibly just one, of the sugar receptors present in the sugar neuron. Worth noting in this context is the fact that electrophysiological recordings are carried out at significantly higher substrate concentrations (up to 100 mM for sucrose and trehalose) than behavioral experiments (2 mM for sucrose and 25 mM for trehalose). The model is also more consistent with recent experiments (Hiroi, 2002) that noted different electrophysiological sugar responses among labellar sensilla (Thorne, 2004).

Approximately 45 labellar neurons have not yet been associated with any Gr gene, and some of these neurons might express putative candidate receptors for sucrose or additional sugars. These genes are likely to be encoded by members of the Gr64 gene cluster, which share much higher sequence similarity with Gr5a than any other Gr genes. Gal4 drivers for two of these genes (Gr64a and Gr64e) were analyzed and found to be expressed in the pharyngeal taste organs, but not in the labellum. Whether these two receptors are indeed involved in sugar detection remains to be seen, but it is predicted that other Gr genes for sugars like sucrose and fructose are be expressed more broadly and in taste neurons of labellar bristles and pegs (Thorne, 2004).

In summary, expression and behavioral studies suggest two fundamentally different roles for neurons expressing nonoverlapping groups of Gr genes in the detection of substrates that lead to feeding or avoidance behavior. According to this new model, S and I bristles on the labial palps contain one avoidance neuron and one or more feeding neurons (depending on the number of neurons associated with the particular bristle). The avoidance neuron expresses multiple Gr genes, and avoidance neurons of different bristles express these Gr genes in different combinations (the Gr gene code). The feeding neurons, which appear morphologically smaller than the avoidance neurons, express an entirely different set of receptors that includes Gr5a and possibly Gr genes encoding receptors for other sugars, amino acids, and peptides (Thorne, 2004).

The different functions for GRNs expressing Gr66a and Gr5a are also supported by their different projection patterns in the brain. Neurons expressing Gr66a or any of the partially coexpressed receptors target similar regions in the SOG/tritocerebrum, though the number of termini differs significantly depending on the number of peripheral sensory neurons the Gr is expressed in. For example, Gr66a-expressing neurons show a robust array of termini in the SOG/tritocerebrum, whereas the termini of Gr59b- and Gr22f-expressing neurons are significantly less numerous. In all cases, dense, contralaterally projecting fibers provide extensive innervation of both halves of the SOG by labellar neurons, as demonstrated by labial palp ablation experiments (Thorne, 2004).

An entirely different projection pattern is observed for feeding neurons that express Gr5a. Most strikingly, the axon termini of these neurons are distributed over a very large area of the SOG and extend into regions not innervated by avoidance neurons. A second striking difference is the poorly established contralateral connective between the two halves of the SOG, suggesting that neurons located in the right labial palp preferentially terminate in the right half of the SOG. This idea was tested and confirmed through ablation studies and has interesting implications, namely that spatially restricted activation of neurons in one palp will preferentially stimulate the same side of the SOG; this could potentially allow for spatial discrimination of taste input in the brain. This feature might allow the fly to orient its labellum in the direction of a food source, identifying regions with high concentrations of trehalose or other sugars (Thorne, 2004).

The distinct pattern of axon termini in the SOG of neurons required for feeding and avoidance suggests that these behaviors are mediated through different neuronal pathways. Anatomical studies in honeybees have identified second-order neurons that mediate synaptic activity of primary taste neurons to higher brain centers. It will be interesting to see whether second-order neurons contacting synapses of avoidance and feeding neurons define different target regions in these higher brain centers (Thorne, 2004).

Taste is an ancient sense, which exists in bacteria in the form of chemotaxis. Neuroanatomical and molecular comparison of taste systems between mammals and insects imply that this sense has evolved independently in these phyla. In mammals, for example, taste ligands are perceived through sensory epithelial cells in the lingual epithelium of the tongue. These cells then activate secondary neurons that innervate taste centers in the brain. In insects, tastants are detected by primary sensory neurons that directly innervate the CNS. Moreover, insects have multiple taste organs (legs, wings, and in some cases, the female genitalia) for which no counterparts exist in mammals. Finally, sequence comparison of the Gr and T1R/T2R genes has failed to reveal any direct kinship between mammalian and insect taste receptors (Thorne, 2004).

However, a remarkable convergence of anatomical as well as molecular features of gustatory systems between mammals and insects (Drosophila) appears to emerge from these studies. The functional taste units, the taste buds in the tongue and the taste bristles of the labellum, are composed of 30 to 100 taste cells and two to four chemosensory neurons, respectively. Individual taste cells in each taste bud are dedicated to the perception of sweet, umami, or bitter taste sensation based on the T1R or T2R receptors they express. Similarly, the data indicate that taste bristles of the labellum contain neurons that either respond to repulsive or attractive stimuli, properties that are likely determined by the specific (set of) taste receptors they express (Thorne, 2004).

Despite the sequence divergence of mammalian and insect taste receptors, it is believed there are intriguing similarities at the molecular level as well. The number of taste receptors in mammals and Drosophila is very similar. The eight genes described in this study probably encode a significant number of the functional labellar taste receptors. Some of the 60 Gr genes are likely to encode taste receptors only expressed in the pharynx, legs, and wings or might only be expressed in the larva. Other Gr genes are likely to function as pheromone receptors, or might recognize internal ligands based on their restricted expression in the CNS. Considering these alternative functions for some Gr genes, it is estimated that the fly has about 30 to 45 labellar taste receptors, a number close to the total number of T1Rs and T2Rs (30 in humans and 45 in mice) (Thorne, 2004).

In addition to the similar size of the Gr and T1R/T2R gene families, taste receptors of mammals and Drosophila fall into similar functional groups. Only three mammalian T1R receptors are thought to be dedicated to the detection of attractive stimuli (sugars and amino acids/proteins), whereas the large majority -- the T2Rs -- are thought to be exclusively involved in the detection of repulsive (bitter) ligands. If the current expression analysis is more or less representative of the entire Gr gene family, it might be expected that 25 to 40 Gr genes will be expressed in the avoidance neurons, whereas just three to six are expected to be expressed in feeding neurons. Identification and analysis of Gr genes encoding receptors for known ligands, combined with biochemical analyses, should reveal whether additional molecular features are shared between the GRs and T1Rs and T2Rs, such as whether Drosophila also possess a specific receptor for amino acids and whether some receptors also function as multimers, as is proposed for mammalian T1Rs (Thorne, 2004).


PROTEIN STRUCTURE

Amino Acids - 392

Structural Domains

Sequence analysis reveals that the putative Tre1 gene, identified in the Ishimoto (2000) study as CG3171, contains 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 the Tre1 cDNA sequence contains seven hydrophobic stretches that represent potential transmembrane domains. 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. The Tre1 gene, as identified by Ishimoto, most closely resembles two other orphan receptors of Drosophila: EG:22E5.11 and EG:22E5.10 (Brody, 2000). It is suggested that the Tre gene may represent a new subclass of taste receptors (Ishimoto, 2000).


Gustatory receptor 5a: Developmental Biology | Regulation | Effects of Mutation | References

date revised: 20 November 2004

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