InteractiveFly: GeneBrief

Gustatory receptor 64a: Biological Overview | References

BIOLOGICAL OVERVIEW

The molecular basis of sugar reception in Drosophila has been analyzed. The response spectrum, concentration dependence, and temporal dynamics of sugar-sensing neurons has been defined. Using in situ hybridization and reporter gene expression, members of the Gr5a-related taste receptor subfamily were identified that are coexpressed in sugar neurons. Neurons expressing reporters of different Gr5a-related genes send overlapping but distinct projections to the brain and thoracic ganglia. Genetic analysis of receptor genes shows that Gr5a is required for response to one subset of sugars and Gr64a for response to a complementary subset. A Gr5a;Gr64a double mutant shows no physiological or behavioral responses to any tested sugar. The simplest interpretation of these results is that Gr5a and Gr64a are each capable of functioning independently of each other within individual sugar neurons and that they are the primary receptors used in the labellum to detect sugars (Dahanukar, 2007).

A major problem in neurobiology is how an animal decides what to eat. The fruit fly evaluates gustatory input to assess the nutritive value of a potential food source. In particular, the detection of sugars is a crucial factor in determining whether a food source is accepted. Despite its critical importance to the survival of the species, little is known about the molecular basis of sugar perception in the fly. A central goal in the field has been to define the receptors that mediate sugar detection (Dahanukar, 2007).

Sugars, salts, bitter compounds, and certain other molecules are detected by gustatory neurons, which are widely distributed in the body of the fly. Neurons that influence feeding behavior are present in the labellum as well as the tarsal segments of each of the legs. Activation of either labellar or tarsal gustatory neurons with a sugar solution results in proboscis extension, which is a component of feeding behavior (Dahanukar, 2007).

Gustatory neurons are housed in sensory hairs called sensilla. Each half of the labellum is covered with ~31 prominent taste hairs, arranged in a stereotypical pattern, and a number of smaller structures called taste pegs. Each of the 31 sensilla is typically innervated by four gustatory neurons and a single mechanosensory neuron. Physiological analysis has shown that one of the chemosensory neurons is activated by sucrose and other sugars, and has been referred to as the 'sugar' neuron. Another neuron is activated by salts and has been named the 'salt' neuron. A third neuron is activated by pure water but not by solutions of high osmolarity; it has been named the 'water' neuron. The fourth chemosensory neuron responds to aversive compounds such as caffeine, and has been named the (Dahanukar, 2007 and references therein).

In Drosophila, a large, highly diverse family of gustatory receptor (Gr) genes was identified by genomic analysis. The family consists of 60 genes encoding 68 predicted seven-transmembrane-domain receptors (Robertson, 2003). In previous studies, Gr5a was identified as a receptor for trehalose, a disaccharide sugar (Chyb, 2003). Gr5a is expressed in a large number of gustatory neurons in the labellum (Chyb, 2003), and recent studies have shown that Gr5a serves as a marker for the sugar neuron in each sensillum (Marella, 2006). Bitter neurons express Gr66a (Thorne, 2004; Wang, 2004), also a member of the Gr gene family, which is required for physiological and behavioral responses to caffeine (Moon, 2006). Promoter expression analysis of several other gustatory receptor genes in the labellum suggested that all of those tested were coexpressed with Gr66a in subsets of bitter neurons (Thorne, 2004; Wang, 2004; Dahanukar, 2007 and references therein).

Axonal projections of Gr5a-positive and Gr66a-positive neurons have been mapped to the subesophageal ganglion (SOG) of the brain (Thorne, 2004; Wang, 2004). The two classes of neurons project to nonoverlapping regions in the SOG, suggesting that at the first level of processing, attractive and aversive inputs may be segregated. Evidence that Gr5a neurons mediate attractive signals and Gr66a neurons mediate aversive signals was provided by expression of a capsaicin receptor in each of these classes of neurons (Marella, 2006). In the first instance, flies showed behavioral attraction to capsaicin, and in the second instance they were repelled by it (Dahanukar, 2007).

Gr5a-labeled neurons are responsive not only to trehalose, but to sucrose and other sugars (Wang, 2004; Marella, 2006). Physiological and behavioral analysis showed that sucrose response is not affected in flies lacking Gr5a, suggesting that these neurons express at least one other receptor; however, other receptors in sugar neurons were not identified (Dahanukar, 2007).

This study examined the responses of sugar neurons in the largest sensilla of the labellum, the 'L' sensilla. Of 50 compounds tested, including 34 diverse sugars, a small number were identified, primarily disaccharides and oligosaccharides, which elicit robust electrophysiological responses in sugar neurons. In situ hybridization and reporter gene expression determined that two other Gr genes, both phylogenetically related to Gr5a, are coexpressed with Gr5a in sugar neurons. Neurons expressing reporters of each receptor gene show distinct projection patterns, providing a mechanism by which information from different subpopulations of sugar cells in the periphery could be spatially represented in the brain (Dahanukar, 2007).

Having found coexpression of Gr5a-related genes in sugar neurons, mutants of Gr5a and two related genes were examined by electrophysiology and behavioral analysis. Gr5a was found to be required for detection of a small subset of sugars including trehalose. Deletion mutants lacking Gr64a shows that it is required for response to a complementary subset of sugars. Strikingly, flies lacking both Gr5a and Gr64a do not show electrophysiological or behavioral responses to any tested sugar. These results demonstrate that the sugars divide into two classes that are dependent either on Gr5a or on Gr64a for their responses. The simplest interpretation of these results is that these two receptors are capable of operating independently of each other in an individual sugar neuron, and that they constitute the primary basis of sugar reception in the fly (Dahanukar, 2007).

Sucrose generated the strongest responses among a panel of 50 compounds tested at 100 mM. Sucrose is present at comparable concentrations in many fruits, including citrus, peaches, and pineapples. Turanose, palatinose, and leucrose are all isomers of sucrose and also elicit responses of various strengths. Many of the sugars that evoke responses, including glucose and trehalose, are found in fruits and vegetables or in yeasts and may thus be encountered by the fly in its natural environment (Dahanukar, 2007).

The responses depend on sugar concentration as well as identity. The neurons are sensitive to a number of sugars over concentrations that span three orders of magnitude. The dose-response curves of different sugars, however, are distinct: they differ in threshold, slope, and maximal firing rate observed. Many of these sugars are present in fruits at concentrations of 100-300 mM, and at these concentrations the responses lie well within the dynamic range of the neurons. Surprisingly, responses to fructose and glucose, which are particularly abundant in fruits, are much weaker than those of sucrose, even when compared at concentrations that have equal caloric values. However, the concentrations of both fructose and glucose are typically higher than that of sucrose in fruits such as apples, bananas, and grapes, suggesting that sugar neurons may be most sensitive to changes in sugar concentrations over a range that is ecologically relevant (Dahanukar, 2007).

Molecular analysis has revealed coexpression of Gr61a and Gr64f with Gr5a, and genetic analysis of a double mutant has provided evidence for coexpression of Gr64a with Gr5a in sugar neurons. These results suggest that at least some labellar sugar neurons, including those of L-type sensilla, coexpress four receptors of the Gr5a subfamily (Dahanukar, 2007).

Molecular and genetic evidence indicates that Gr5a is expressed in essentially all labellar sensilla. Molecular analysis has provided evidence that Gr64f is also broadly expressed, and functional evidence suggests that Gr64a is as well. Specifically, an electrophysiological survey showed that all labellar sensilla in wild-type flies respond to sucrose, a sugar that acts via Gr64a. In a Gr5a;Gr64a mutant all morphological types of sensillum (L, M, S, I, P) showed no activity in response to sucrose; moreover, nearly all of the L, M, I, and P sensilla were tested, suggesting that Gr64a acts in all, or almost all, of the 31 sensilla on the labellum. Furthermore, the double mutants are also behaviorally unresponsive to sugars. Thus Gr5a and Gr64a seem likely to be expressed in all or almost all sugar neurons in the labellum, and perhaps Gr64f is as well (Dahanukar, 2007).

Gr61a, however, appeared to be restricted in its expression among labellar sensilla, both by in situ hybridization and by analysis of a Gr61a-GAL4 driver. These results suggest a subdivision of labellar sugar neurons into two classes based on the presence or absence of Gr61a. No function was defined for Gr61a; however, mutational analysis suggests that it does not play a role in responses to any of the sugars in the panel. It is possible that Gr61a is required for response to other sugars or sugar derivatives that have not yet been tested or for responses to another class of behaviorally attractive compounds. Further electrophysiological analysis with an expanded panel of tastants may provide insight into whether there are functional differences among sugar-sensing neurons and whether these differences correlate with the expression of Gr61a (Dahanukar, 2007).

Gr5a and Gr64a are both required for normal responses of sugar neurons, but for different subsets of sugars. Flies lacking Gr5a are severely defective in physiological and behavioral responses to one subset of sugars, including trehalose; flies lacking Gr64a are severely defective in responses to a complementary subset of sugars, including sucrose. All tested sugars fall into one of these two subsets. These results suggest that Gr5a and Gr64a function as distinct receptors in the same neurons, rather than as obligate heterodimeric coreceptors, as in the mammalian sugar receptor T1R2+T1R3 (Dahanukar, 2007).

It is possible that Gr5a and Gr64a function as heterodimeric receptors with other members of the Gr family, such as Gr64f. Two recent studies report deletions of part or all of the Gr64 cluster that result in reduced behavioral responses to trehalose; the phenotype is rescued by supplying a transgene containing five of the six receptors encoded by this cluster (Slone, 2007), but not by Gr64a alone (Jiao, 2007). These data support the idea that one of the receptors in this cluster other than Gr64a may function in concert with Gr5a to mediate trehalose response. There is precedent for such interactions from Or proteins, which dimerize with the noncanonical receptor Or83b (Dahanukar, 2007).

The neat subdivision of sugars into those dependent on Gr5a and those dependent on Gr64a was surprising. A simple structural criterion to distinguish the two classes of sugars is not immediately evident upon inspection. The Gr64a-dependent sugars are remarkably diverse in structure, with some containing glucose units and some containing fructose subunits; they ranged in size from one to four subunits. Gr5a-dependent sugars also vary in size, subunit composition, and linkage types (Dahanukar, 2007).

In Gr5a mutants, there are some weak residual responses to the affected subset of sugars; likewise, in Gr64a mutants, some of the affected sugars continue to elicit some response. Since there is no residual response in the Gr5a;Gr64a double mutant, the simplest interpretation of these results is that each receptor provides the residual function observed when the other is eliminated, i.e., the two receptors exhibit some limited redundancy (Dahanukar, 2007).

Gr5a and Gr64a share 28% amino acid identity and 47% amino acid similarity. Both receptors are evolutionary conserved and are found in all of the 12 Drosophila species for which genome sequences are available, with the exception that D. pseudoobscura appears to have lost Gr5a. The receptor most closely related to Gr5a is Gr64f (40% amino acid identity), and the receptor most closely related to Gr64a is Gr61a (36% amino acid identity). Although evidence was found that Gr64f and Gr61a are both expressed in sugar neurons, no functions have been identifed for them. The possibility cannot be excluded of a role for Gr61a or Gr64f in response to compounds not tested, such as glycoproteins or glycolipids, or in neurons whose responses have not been measured, such as those of internal chemosensory cells. It is noted that in mammals, an amino acid receptor (T1R1+T1R3) comprises a subunit, T1R3, of the heterodimeric sugar receptor (T1R2+T1R3) (Nelson, 2001; Nelson, 2002). However, L-type sensilla did not respond to any of 18 amino acids tested, making it unlikely that either Gr61a or Gr64f mediates responses to this class of compounds (Dahanukar, 2007).

Classic physiological and biochemical studies led to the proposal of a 'fructose' site in sugar-sensing neurons. The current studies provide a molecular and genetic identity to this site: fructose response is completely abolished by loss of Gr64a and is completely restored by the addition of a Gr64a transgene. These results also provide a molecular explanation for the earlier finding that sucrose responses were not affected in a Gr5a mutant. These results suggested the presence of another receptor within the sugar neuron, a receptor that has now been identified as Gr64a (Dahanukar, 2007).

It is noted that two recent studies have identified a role for members of the Gr64 cluster in mediating sugar responses (Jiao, 2007; Slone, 2007), particularly that of Gr64a in response to sugars including sucrose, maltose, and glucose (Jiao, 2007). Consistent with the observations, physiological and behavioral responses to sucrose were restored to wild-type levels in transgenic rescue experiments; no role was observed for Gr64a in glucose response. One of these studies (Jiao, 2007) also provided biochemical evidence that Gr5a-related receptors are expressed in sugar-sensitive neurons (Dahanukar, 2007).

In summary, the simplest interpretation of the results is that Gr5a and Gr64a are the primary sugar receptors in the labellum of the adult fly. Each is capable of mediating response to a subset of sugars independently of the other, and together they are able to identify the food sources that are sufficiently rich in caloric value as to sustain the life of the fly (Dahanukar, 2007).

A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging

In Drosophila, detection of tastants is thought to be mediated by members of a family of 68 gustatory receptors (Grs). However, only one receptor, Gr5a, has been associated with a sugar, and it appears to be activated specifically by trehalose. It is unclear whether other sugar receptors are activated by single or multiple sugars. Currently, no Grs are known to colocalize with Gr5a. Such Grs would be candidate sugar receptors because Gr5a-expressing cells function in the responses to attractive tastants. This study used an 'mRNA tagging' approach to identify Gr RNAs that are coexpressed with Gr5a. All seven Grs most related to Gr5a (Gr64a-f and Gr61a) are expressed in Gr5a-expressing cells, whereas none of the other Grs examined were enriched in these Gr neurons (GRNs). The role of one Gr5a-related receptor, Gr64a, was characterized and it was found that Gr64a is required for the behavioral responses to glucose, sucrose, and maltose. Gr64a is required for GRN function because action potentials induced by these sugars were dependent on expression of Gr64a in GRNs. These data demonstrate that multiple Grs are coexpressed with Gr5a and that Drosophila Gr64a is required for the responses to multiple sugars (Jiao, 2007).

To identify Grs that were candidate sugar receptors, tests were performed to see whether any Gr RNAs were coexpressed with Gr5a. Given that in situ hybridizations to Gr RNAs have been problematic for most Gr genes, an mRNA tagging approach was used in combination with the GAL4/UAS system. The mRNA tagging approach entails purification of RNAs from specific cell populations using a FLAG-tagged poly(A)-binding protein (PABP). To screen for Grs expressed in Gr5a GRNs, extracts were prepared from dissected labella expressing Gr5a-GAL4;UAS-PABP or Gr66a-GAL4;UAS-PABP transgenes, then immunoprecipated the FLAG-PABP with anti-FLAG antibodies and the mRNAs were isolated that were pulled down with the PABP (Jiao, 2007).

To test the efficacy of the mRNA tagging, RT-PCR was performed using primers specific for Gr5a and Gr66a. As a control, RT-PCR was performed using primers specific for a pan-neuronally expressed gene, elav, which was therefore detected in both Gr5a- and Gr66a-expressing GRNs. In contrast to these results, the Gr5a signal was much higher using RNA prepared from Gr5a-GAL4;UAS-PABP than from Gr66a-GAL4;UAS-PABP flies. Conversely, the Gr66a product was found primarily by using RNA from Gr66a-GAL4;UAS-PABP flies. These results indicated that the mRNA tagging approach may be an effective assay to examine whether other Grs are expressed in either Gr5a- or Gr66a-expressing GRNs (Jiao, 2007).

Initially the expression of a set of eight Grs was surveyed that were distributed among a variety of branches within the Gr family tree. Two of the Gr RNAs (Gr22e and Gr32a) were predicted to be enriched in Gr66a-positive neurons because the corresponding GAL4 reporters have been shown to be expressed in subsets of Gr66a GRNs. In addition, Gr63a expression, which was unlikely to be coexpressed with either Gr5a or Gr66a was tested because this Gr encodes a CO₂ receptor. Gr64b belongs to a distinct branch that includes the seven Grs most related to Gr5a (28-45% amino acid identities; referred to here as the Gr-S group) (Jiao, 2007).

It was found that Gr64b was the only one among the eight surveyed that was enriched in Gr5a-GAL4;UAS-PABP flies. In contrast, five of the Gr RNAs were found primarily in the RNA prepared from Gr66a-GAL4;UAS-PABP, including three whose expression had not been previously characterized (Gr33a, Gr39aD, and Gr59f). No Gr63a product was detected in either RNA sample, which was expected because Gr63a is a CO₂ receptor. A Gr98a RT-PCR band also was not detected, which might be because of its low expression level (Jiao, 2007).

Because Gr64b was among the group of seven Grs most related to Gr5a, whether the remaining six members of the Gr-S group were enriched in Gr5a-GAL4;UAS-PABP flies was tested. It was found that the RT-PCR products of all Gr-S RNAs (Gr61a and Gr64a-f) were expressed predominately in Gr5a-GAL4;UAS-PABP flies. These included Gr64a and Gr64e, despite the report that Gr64a- and Gr64e-GAL4 reporter expression was not detected in the labellum. Thus, of the 14 Grs tested, all seven Gr-S RNAs but none of the other Grs were enriched in Gr5a-GAL4;UAS-PABP flies (Jiao, 2007).

To test the proposal that Gr-S receptors other than Gr5a are sugar receptors, the requirement for Gr64a was tested. An additional question is whether other Drosophila sugar receptors are activated by one or multiple sugars. In contrast to the mammalian sugar receptors, Gr5a was reported to be specifically activated by trehalose. This observation, in combination with the larger number of candidate sugar receptors in flies than mammals, raised the possibility that each Gr-S member may respond primarily to one sugar. However, it was found that the Gr64a gene was required in vivo for the responses to the monosaccharide, glucose, and the disaccharides, sucrose and maltose, each of which includes at least one glucose subunit. Gr64a was not essential for the responses to all sugars, because the Gr64^ab flies responded normally to the monosaccharides, fructose and arabinose. Although the defects in the behavioral responses to sucrose, glucose, and maltose were completely rescued by expression of a wild-type Gr64a transgene, only the sucrose response was fully rescued as assayed by tip recordings. The rescues of the electrophysiological responses to glucose and maltose were significant but did not restore the same frequencies of action potentials as in wild type. Although the explanation for this result is unclear, similar findings were reported for rescue of the trehalose deficits in the Gr5a^Delta5 allele by a wild-type transgene. Whereas the behavioral phenotype in Gr5a^Delta5 was restored entirely, the rescue of the electrophysiological response to trehalose was partial (Jiao, 2007).

The trehalose response was also greatly reduced in Gr64^ab mutant flies. This result was unexpected because the response to this sugar is nearly eliminated in the Gr5a mutant, and Gr5a is sufficient to confer trehalose sensitivity in S2 cells. Nevertheless, there is small residual trehalose response in the Gr5a mutant. The trehalose defect in Gr64^ab flies did not appear to arise from a background mutation in Gr5a, because the phenotype was observed in flies in which the Gr64^ab deletion was placed in trans with deficiencies that spanned the Gr64 locus. Thus, the question arises as to the identity of the second trehalose receptor. The deletion in Gr64^ab disrupts both Gr64a and Gr64b, and introduction of a wild-type UAS-Gr64a transgene under the control of the Gr5a-GAL4 restores normal responses to sucrose, glucose, and maltose, but not to trehalose. Thus, Gr64b may be a trehalose receptor. However, a UAS-Gr64b transgene alone or in combination with the UAS-Gr64a failed to restore a trehalose response in flies containing the Gr5a-GAL4, suggesting that either Gr64b is not a trehalose receptor or the transgene is nonfunctional (Jiao, 2007).

A general issue concerning the Drosophila Grs is whether they typically form homo- or heteromultimers. An indication that at least some Grs form obligatory heteromultimers is that misexpression of just one of the two CO₂ receptor genes, Gr21a or Gr63a, in CO₂-insensitive antennal neurons is insufficient to confer CO₂ sensitivity to these cells. However, coexpression of both Gr21a and Gr63a induces CO₂ responsiveness. Misexpression of just Gr64a in Gr66a GRNs did not elicit an aversive response to sucrose, glucose, or maltose or result in sugar-induced action potentials in Gr66a GRNs. Similarly, expression of the caffeine receptor Gr66a in Gr5a-expressing cells does not confer caffeine sensitivity to these cells. These results raise the possibility that these and possibly other taste receptors in flies are obligatory heterodimers, as is the case for the CO₂ receptors. The other Gr-S members would appear to be the best candidates for forming heteromultimers with Gr64a. Finally, it is proposed that the mRNA tagging approach applied here can be extended to identify pairs of Grs that are expressed together in smaller subsets of GRNs and would therefore be excellent candidates for forming heteromultimers (Jiao, 2007).

Sugar receptors in Drosophila

Detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly non-volatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor, the functions of GRs involved in feeding are unknown. This study shows that the Gr64 genes encode receptors for numerous sugars. A fly strain was generated that contained a deletion for all six Gr64 genes (ΔGr64); these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, evidence is presented that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for co-expression of multiple sugar receptors in the same taste neurons (Slone, 2007).

To gain a basic understanding of sugar perception in Drosophila, a reverse genetic analysis of the six Gr64 genes, which are tightly clustered on the left arm of chromosome 3, was performed. FRT mediated trans-recombination was used to create a 25 kb deletion of the region containing the Gr64a gene cluster (ΔGr64), and the expected molecular nature of this deletion was confirmed using genomic PCR and DNA sequencing from the trans-recombined chromosome. In addition to the six Gr64 genes, this trans-recombination event also removed five additional genes on either side of the cluster, resulting in a homozygous lethal mutation, presumably because some of the neighboring genes have essential functions required for viability. Therefore two genomic DNA constructs were cloned containing the two genes proximal (R1) or the three genes distal (R2) to the Gr64 locus, respectively, into a transformation vector, and corresponding transgenic Drosophila lines were generated. When R1 was crossed into the ΔGr64 mutant strain, viability was completely restored, indicating that at least one of the two proximally located genes provides a life-essential function. Even though unlikely, it is possible that some genes on R1 and/or R2 may have functions related to taste perception, ΔGr64/ΔGr64 flies that carried a copy of each of these rescue constructs were used for all behavioral experiments (Slone, 2007).

Whether the Gr64 genes were required for the detection of six sugars was tested by generating ΔGr64 homozygous mutant flies that contained one copy of each of the rescue constructs (R1/+;R2/+;ΔGr64/ΔGr64). The behavioral response of these and control flies was determined to sucrose, glucose, trehalose, fructose, arabinose and maltose using the proboscis extension reflex (PER). As controls, flies were tested that were heterozygous for each of the two piggyBac elements used to generate the ΔGr64 mutation, as well as flies with an intact Gr64 cluster, but containing a copy of R1 and R2 to rule out a dominant phenotype of these transgenes. PER is a robust indicator of a fly’s attraction and motivation to eat a given chemical compound. If taste neurons in labial palps or the forelegs are stimulated with a solution containing sugars, the fly extends its proboscis to attempt feeding. Indeed, it was found that both control strains responded with high probability of a PER, ranging from 42% to 97% when stimulated with 500 mM solution of various sugars. Even at a five-fold lower concentration (100 mM), both types of control flies responded to all sugars, albeit with a reduced PER. In contrast, R1/+;R2/+;ΔGr64/ΔGr64 flies showed a drastic reduction in PER for all sugars at both 500 and 100 mM, except for fructose. In most cases, the reduction was at least 10 fold, while sensitivity for sucrose was reduced only by about three-fold. However, PER response to fructose was the same in control flies and R1/+;R2/+;ΔGr64/ΔGr64 mutant flies at 100mM and reduced by only about 35% at 500mM, suggesting that a high affinity fructose receptor is present in flies lacking all six Gr64 genes (Slone, 2007).

It has been recently shown that flies exhibit a behavioral feeding response to glycerol, a linear triol, and indeed, glycerol was shown to stimulate sugar-sensitive neurons. It was asked whether glycerol detection is also mediated by some of the GR64 receptors, and therefore the PER response was examined in control and ΔGr64 mutant flies. Indeed almost a 6 and 16 fold reduction of PER to 2% and 10% glycerol, respectively, was observed in mutants when compared to controls (Slone, 2007).

The loss of behavioral response to trehalose in ΔGr64 mutant flies is surprising, since these flies contain presumably a wild type Gr5a gene, which encodes a receptor for this sugar. Therefore, the perception of trehalose appears to require at least two Gr genes, Gr5a and one or more members of the Gr64 gene cluster, suggesting that insect sugar receptors might function as dimers or multimers. To rule out the possibility that the loss of trehalose responses in these flies is caused by a defective Gr5a gene, the PER response was tested of flies heterozygous for ΔGr64, but containing the same X chromosome (i.e. the same Gr5a) as the homozygous ΔGr64 flies. These flies showed indeed a robust response to trehalose, indicating that a second receptor in the Gr64 locus is necessary for the detection of this sugar. Thus, trehalose, and possibly sugars in general, are detected by mulimeric receptors composed of two or more GRs (Slone, 2007).

Homozygous ΔGr64 mutant flies show as robust a response to 100 mM fructose as control flies, indicating that a functional fructose receptor does not contain any of the GR64 proteins. A receptor for this sugar might therefore be comprised of a heterodimer between GR61a and GR5a or a homodimer of either one of these two proteins. But other compositions are possible as well, such as heterodimers involving one of these subunits along with another GR proteins. Any such dimer may also serve as a low affinity receptor for non-fructose sugars and therefore be responsible for the residual PER responses to glucose, trehalose, maltose and arabinose in homozygous ΔGr64 mutant flies (Slone, 2007).

To assess whether lack of the Gr64 gene affects the behavioral responses to other chemicals, PER response was tested to four chemically diverse, bitter-tasting compounds. Such compounds, which are known to inhibit feeding, reduce PER responses if they are mixed with sugars solutions. Therefore, PER responses were tested to 500 mM fructose solutions that included caffeine, denatonium benzoate, berberine or quinine. Both strains showed a similar decrease in PER response when stimulated with these solutions, suggesting that the Gr64 genes are not required for the detection (and avoidance) of bitter compounds. Taken together, these data suggest that the six Gr64 genes are necessary specifically for the detection of most sugars (Slone, 2007).

The Gal4/UAS expression system has been used very successfully to identify GRNs that express specific Gr genes. Four Gr64-Gal4 driver constructs were generated and combined with the UAS-gfp reporters, but no expression was observed in the main taste organs with any of them, even though RNAs for all six Gr64 genes are detected by RT-PCR. This suggested that crucial transcriptional regulatory elements are located upstream and/or downstream of the cluster and/or within introns of the Gr64 genes. Further support for an unusual arrangement of regulatory elements of the Gr64a genes is apparent from the dense genomic clustering of the six open reading frames (ORFs). Assuming at least 50 nucleotides of 5' and 3' UTR for each gene, the intergenic, non-transcribed regions harboring putative promoters are extremely short (<100 nt) and lack transcription termination signals (AAUAAA), which are present in most Drosophila genes. These observations prompted a test of whether the Gr64 genes might be transcribed as a poly-cistronic mRNA. mRNA was isolated from heads and RT-PCR analysis was performed across the whole cluster using primer pairs of adjacent genes. To discriminate between products from spliced RNA and residual genomic DNA, primers were chosen such that the amplified fragments would represent spliced transcripts that lack at least one intron. In each case, RT-PCR readily amplified a spliced RNA product composed of cDNAs corresponding to adjacent ORFs separated by the intergenic sequence. The same result was obtained when RNA isolated from leg tissue was used. These results suggest that coding sequences of adjacent Gr64 genes are present on the same mRNA and, by inference, that possibly all six ORFs may be transcribed as a large polycistronic mRNA (Slone, 2007).

With the exception of nematodes, polycistronic transcripts are not thought to be common in higher eukaryotes. In C. elegans, however, a significant number of genes (~15 %) are co-transcribed as operons, and independently trans-spliced to the abundantly expressed spliced leader (SL2) RNA. However, it has recently become apparent that operon-like gene organizations and polycistronic mRNAs do exist in Drosophila; at least two transcripts initially postulated to be non-coding RNAs were shown to encode multiple, albeit redundant peptides, with functions necessary in early development. Examples more similar to the Gr64 genes were described for four pairs of Drosophila Or genes and the Drosophila CheB42a-llz locus. The basic translation mechanism of poly-cistronic mRNAs of the Or gene pairs is unknown; however the CheB42a-llz dicistronic transcript is subsequently cleaved into two mRNAs that appear to be translated separately (Ben-Shahar, 2007). While a polyadenylation signal is present after the upstream gene (CheB42a) in this case, no putative promoter sequences were identified for the intergenic region in the CheB42a-llz locus. A genomic survey by the authors of this paper, for closely clustered genes lacking promoter sequences in the intergenic region, identified almost 1400 Drosophila gene pairs, suggesting that operon-like gene structures may be much more common in eukaryotes than generally assumed. Not surprisingly several Or and Gr gene pairs were found to lack such promoter sequences, including the five downstream genes in the Gr64 gene cluster (Slone, 2007).

To prove conclusively that the Gr64 genes indeed encode sugar receptors, transgene rescue experiments were performed. A genomic fragment containing five of the six Gr64 genes was cloned into the UAS reporter (UAS-Gr64abcd_GFP_f; Gr64e was replaced by GFP), and two types of R1/+;R2/+;ΔGr64/ΔGr64 flies were generated, the first containing the UAS-Gr64abcd_GFP_f rescue construct, and the second containing the same rescue construct as well as Gr5a-Gal4 driver. The Gr5a-Gal4 driver is expressed in sugar-sensitive neurons of both the labellum and the legs and should confer such expression on the rescue construct. At both 100mM and 500mM concentration, the UAS-Gr64abcd_GFP_f reporter rescued the PER response to similar levels as observed in the control strain. Surprisingly, this rescue was independent of the GAL4 driver, indicating that intragenic regulatory elements confer sufficient expression onto the Gr64 genes. This expression was confirmed using RT-PCR analysis, which showed that regardless of whether the Gr5a-Gal4 was present or not, the Gr64 transcripts were readily amplified. It is noted that a second Gr64abcd_GFP_f reporter integrated in a different genomic location provided only partial rescue (Slone, 2007).

This study has shown that the six Gr64 genes encode receptors for the detection of most sugars: sucrose, glucose, maltose, trehalose, and arabinose. The data also suggests that the Drosophila taste receptors, similar to insect olfactory and CO₂ receptors and mammalian sweet taste receptors, function as dimers (or possibly multimers), since detection of trehalose requires, in addition to GR5a, at least one of the six receptors encoded by the Gr64 genes. However, in contrast to mammals, which use T1R2/T1R3 heterodimers for the detection of all sugars, Drosophila appears to use distinct combinations of GRs for the detection of different sugars. Since GRs have been difficult to express in cell or heterologous systems, the availability of a mutant lacking the six Gr64 genes should help elucidate the molecular nature of dimeric (or multimeric) sugar receptors in insects (Slone, 2007).

RT-PCR analysis of Gr64 transcripts suggests that the six Gr64 genes are transcribed as a polycistronic mRNA, a rare mode of gene expression in eukaryotes other than C. elegans. Of the few known examples of operon-like genes in Drosophila, the CheB42a-llz locus is the best-characterized case of a polycistronic mRNA. The CheB42a-llz RNA, which encodes two proteins, is subsequently cleaved into two transcripts, which appear to be translated independently by a cap-independent process. Elucidating how the Gr64 transcripts are processed post-transcriptionally and by which mechanism they are translated will be a challenging undertaking, especially if such processing only takes place in the correct cellular context (i.e. taste neurons). In any case, polycistronic, operon-like transcription of the Gr64 genes would provide an elegant solution for their coordinated expression in the same subset of sugar-responsive taste neurons (Slone, 2007).

Taste-independent detection of the caloric content of sugar in Drosophila

Feeding behavior is influenced primarily by two factors: nutritional needs and food palatability. However, the role of food deprivation and metabolic needs in the selection of appropriate food is poorly understood. This study shows that the fruit fly selects calorie-rich foods following prolonged food deprivation in the absence of taste-receptor signaling. Flies mutant for the sugar receptors Gr5a and Gr64a cannot detect the taste of sugar, but still consumed sugar over plain agar after 15 h of starvation. Similarly, pox-neuro mutants that are insensitive to the taste of sugar preferentially consumed sugar over plain agar upon starvation. Moreover, when given a choice between metabolizable sugar (sucrose or D-glucose) and nonmetabolizable (zero-calorie) sugar (sucralose or L-glucose), starved Gr5a; Gr64a double mutants preferred metabolizable sugars. These findings suggest the existence of a taste-independent metabolic sensor that functions in food selection. The preference for calorie-rich food correlates with a decrease in the two main hemolymph sugars, trehalose and glucose, and in glycogen stores, indicating that this sensor is triggered when the internal energy sources are depleted. Thus, the need to replenish depleted energy stores during periods of starvation may be met through the activity of a taste-independent metabolic sensing pathway (Dus, 2011).

This taste-independent sugar-sensing pathway has several distinctive characteristics. First, this pathway is specifically associated with a starved state; taste-blind flies execute food-choice behavior after prolonged food deprivation of between 10 and 15 h of starvation. This time frame coincides with the onset of starvation-induced sleep suppression, indicating that these two behaviors might share a common metabolic trigger. Second, the taste-independent pathway operates on a different timescale from the gustatory pathway. Whereas WT flies made a food choice almost instantly, taste-blind flies chose sugars only after the ingestion of food. Third, this pathway responds to the nutritional content of sugars, but not to their orosensory value. Taste-blind flies chose metabolizable sugars over nonmetabolizable sugars and never consumed nonmetabolizable sugars. Furthermore, the fact that WT flies failed to distinguish a metabolizable sugar from a nonmetabolizable sugar, but shifted their preference to the metabolizable sugar after starvation, indicates that the taste-independent pathway is not an artifact associated with taste-blind flies, but functions in WT flies. Finally, the ability to detect the caloric content of sugars correlated under multiple experimental conditions with drops in hemolymph glycemia (Dus, 2011).

These results demonstrate that starvation directs the selection of nutrient-rich foods in the fly in the absence of the gustatory cues. Thus, as previously suggested in mice, postingestive cues can drive feeding behavior independently of gustatory information. The physiological factors that triggered the taste-independent food choices in mice are, however, unknown. In Drosophila, the internal energy state and carbohydrate metabolism play crucial roles in the metabolic sensing of food according to the results. A possible evolutionary purpose of taste-independent metabolic sensing is to ensure that animals select calorie-rich foods to quickly replenish energy, especially in times of food shortage (Dus, 2011).

How do starved sugar-blind flies preferentially ingest metabolizable sugar over nonmetabolizable sugar? It is plausible that sugar-blind flies are equally attracted to and feed on both sugars, but those on nonmetabolizable sugar resume foraging because of the lack of nutritional value in this sugar. These foraging flies are again equally attracted to both sugars, but those on nonmetabolizable sugar continue to forage until they find the correct food substrate. Food choice in this model is mediated by random selection and 'trapping' of the flies on the metabolizable sugar. Alternatively, sugar-blind flies might readily detect the metabolizable sugar without ingesting a large amount of food because nutrient information is rapidly conveyed to the brain within minutes of ingesting food. In this model, the flies select for metabolizable sugar over nonmetabolizable sugar by a metabolic sensor that operates on a fast timescale to mediate discrimination between the two sugar substrates. Tracking and monitoring the locomotor activity and feeding behavior that generates a preference for metabolizable sugar will address this question (Dus, 2011).

It is intriguing to speculate on the molecular nature of the metabolic sensor. This sensor could be expressed in a subset of neural, digestive, or other tissues. Among the organs and cells that have been proposed for their involvement in feeding regulation in the fly are the fat body, the insulin-producing cells (IPC), and the corpora cardiaca/allata complex. These cells may respond to the metabolic value of sugars in circulation, as seen with the glucose-excited and glucose-inhibited neuropeptide neurons in the arcuate nucleus of the mammalian hypothalamus. A model that explains how changes in circulating glucose levels alter the electrical and secretory properties of the hypothalamic glucose-responsive neurons could also describe how metabolizable sugars trigger the metabolic sensor. In mammals, glucose-sensitive cells detect glucose availability by responding to metabolites of glycolytic enzymes such as hexokinase or the energy-sensing AMP-activated protein kinase (Dus, 2011).

Almost all crucial metabolic functions in mammals are also conserved in Drosophila. During the past decade, researchers using the fruit fly as a model system for studying feeding behaviors and feeding-related disorders, including obesity, have shed much light on the molecular mechanisms of metabolism. By revealing the possibility of a metabolic sensing pathway in Drosophila, this study has introduced the possibility of understanding the molecular mechanism underlying this pathway. Identification of the cellular and genetic nature of this sensor might reveal the identity of the master switch that regulates many hunger-driven behaviors (Dus, 2011).

Search PubMed for articles about Drosophila Gr64a

Ben-Shahar, Y., Nannapaneni, K., Casavant, T. L., Scheetz, T. E. and Welsh, M. J. (2007). Eukaryotic operon-like transcription of functionally related genes in Drosophila. Proc. Natl. Acad. Sci. 104: 222-227. PubMed ID: 17190802

Chyb, S., Dahanukar, A., Wickens, A. and Carlson, J.R. (2003). Drosophila Gr5a encodes a taste receptor tuned to trehalose. Proc. Natl. Acad. Sci. 100: 14526-14530. 14523229

Dahanukar, A., Lei, Y. T., Kwon, J. Y. and Carlson, J. R. (2007). Two Gr genes underlie sugar reception in Drosophila. Neuron 56(3): 503-16. PubMed ID: 17988633

Dus, M., et al. (2011). Taste-independent detection of the caloric content of sugar in Drosophila. Proc. Natl. Acad. Sci. 108(28): 11644-9. PubMed ID: 21709242

Jiao, Y., Moon, S. J. and Montell, C. (2007). A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging. Proc. Natl. Acad. Sci. 104(35): 14110-5. PubMed ID: 17715294

Marella, S., Fischler, W., Kong, P., Asgarian, S., Rueckert, E. and Scott, K. (2006). Imaging taste responses in the fly brain reveals a functional map of taste category and behavior. Neuron 49(2): 285-95. 16423701

Moon, S. J., Kottgen, M., Jiao, Y., Xu, H. and Montell, C. (2006). A taste receptor required for the caffeine response in vivo. Curr. Biol. 16: 1812-1817. PubMed ID: 16979558

Nelson, G., et al. (2001). Mammalian sweet taste receptors. Cell 106: 381-390. PubMed ID: 11509186

Nelson, G., et al. (2002). An amino-acid taste receptor. Nature 416: 199-202. PubMed ID: 11894099

Robertson, H. M., Warr, C. G. and Carlson, J. R. (2003). Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster. Proc. Natl. Acad. Sci. 100: 14537-14542. PubMed ID: 14608037

Slone, J., Daniels, J. and Amrein, H. (2007). Sugar receptors in Drosophila. Curr. Biol. 17(20): 1809-16. PubMed ID: 17919910

Thorne, N., Chromey, C., Bray, S. and Amrein, H. (2004). Taste perception and coding in Drosophila. Curr. Biol. 14: 1065-1079. 15202999

Wang, Z., Singhvi, A., Kong, P. and Scott, K. (2004). Taste representations in the Drosophila brain. Cell 117(7): 981-91. 15210117

date revised: 15 December 2011

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.