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Colloquium
Drosophila Gr5a encodes a taste receptor tuned
to trehalose
Sylwester Chyb*, Anupama Dahanukart, Andrew Wickens*, and John R. Carlsont:
*Imperial College London, Wye Campus, Kent TN25 5AH, United Kingdom; and "Department of Molecular, Cellular, and Developmental Biology,
Yale University, New Haven, CT 06520
Recent studies have suggested that Drosophila taste receptors are
encoded by a family of G protein-coupled receptor genes compris-
ing at least 56 members. One of these genes, Gr5a, has been shown
by genetic analysis to be required by the fly for behavioral and
sensory responses to a sugar, trehalose. Here, we show that GrSa
is expressed in neurons of taste sensilla located on the labellum
and legs. Expression is observed in most if not all labellar sensilla
and suggests that many taste neurons express more than one
receptor. We demonstrate by heterologous expression in a Dro-
sophila S2 cell line that Gr5a encodes a receptor tuned to trehalose.
This is the first functional expression of an invertebrate taste
receptor.
The majority of taste sensilla in Drosophila are located on the
labellum, a gustatory organ of the proboscis, and the tarsal
segments of the legs. Most sensilla house four gustatory neurons,
one sensitive to sugars, one strongly sensitive to salt, one weakly
sensitive to salt, and one responsive to water, as well as one
mechanosensory neuron (1, 2~. Previous work has identified a
large family of G protein-coupled receptor genes, the Gr genes,
which have been proposed to encode gustatory receptors in
Drosophila (3~. The Gr family comprises at least 56 members,
many of which are expressed in subsets of taste neurons in the
different taste organs (3-54.
Perception of sugars is a critical taste modality in animals such
as fruit flies that ingest sweet substances for nutrition. Among
the sugars, one that plays a particularly important role in insects
is the disaccharide trehalose ~l-O-(cx-D-glucopyranosyl-~-D-
glucopyranose)], which is composed of two glucose molecules
connected by an unusual 1,1' linkage. Also called mycose, this
disaccharide is abundant in yeasts and fungi, which are present
in fermented fruit, an important food source of Drosophila.
Trehalose is especially critical to insects in that it is the principal
sugar found in the hemolymph, where it is involved in regulation
of osmolarity, and in many winged insects it is an easily trans-
ported and accessible energy source metabolized during
flight (6).
Previous genetic studies have identified a locus on the X
chromosome called Tre, whose alleles confer differing levels of
sensitivity to trehalose (7, 8~. Recently we have shown that a
member of the Gr family, GrSa, maps to this locus and is
necessary for trehalose response in viva (~9~. Deletion mutants of
GrSa have a greatly diminished response to trehalose when
assayed by electrophysiological recordings from single taste
sensilla, or by a behavioral test. This defect was rescued by
reintroducing a functional copy of GrSa on a transgene, but not
by introducing a mutant copy of GrSa. Consistent with our
results, Ueno et al. (10) showed that polymorphisms in Gr5a
correlate with trehalose responses. However, these results do not
exclude the possibility that GrSa plays an indirect role in
trehalose response.
Here we show that GrSa is expressed in taste neurons of the
labellum and the tars), supporting its identity as a taste receptor
14526-14530 1 PNAS 1 November 25, 2003 1 vol. 100 1 suppl. 2
gene. We then provide direct evidence that GrSa encodes a
trehalose receptor by expressing it in Drosophila S2 cells and
showing that stimulation with trehalose evokes changes in
intracellular calcium (Ca2+) levels in these cells. We further show
that GrSa is narrowly tuned to trehalose, showing little if any
response to other disaccharides.
Methods
Expression Analysis. An 8.5-kb fragment upstream of GrSa was
amplified from genomic DNA of Canton-S flies and inserted
upstream of the GAL4-coding sequence in the pG4PN vector (C.
Warr and J.R.C., unpublished data). Transgenic flies were
generated by using standard procedures. Heterozygous flies
carrying one copy each of GrSa-GAL4 and UAS-lacZ were
stained for LacZ activity. For visualization of GFP, flies carrying
two copies of GrSa-GAL4, as well as two copies of UAS-
mCD8:GFP, were examined by using confocal microscopy.
Heterologous Expression. A 1.2-kb fragment of full-length GrSa
cDNA was amplified from head mRNA by using the Smart
RACE cDNA Amplification kit (Clontech). This fragment was
inserted into a unique EcoRI site in the pRmHa3 vector (11~.
Drosophila S2 cells were grown at 25°C in Shields and Sang M3
insect medium supplemented with 10% FCS and antibiotic/
antimycotic solution. S2 cells were cotransfected with
pRmHa3-GrSa and pIZT-V5/His vector (Invitrogen) encod-
ing GFP and zeocin-resistance to facilitate recognition of
transfectants and antibiotic selection, at a 3:1 ratio by using
liposomal formulation (CellFectin, Invitrogen) according to
the manufacturer's instructions. Expression of GrSa was in-
duced by adding 0.6 mM Cu2+ to the cell culture media 48 hrs
before an experiment. Levels of GrSa expression in uninduced
and induced cells were examined by RT-PCR to confirm the
induction of Gr5a.
Calcium Imaging. Transfected cells were grown in 96-well plates
and loaded with membrane-permeable fura 2-acetoxymethyl
ester (fura 2-AM) as described elsewhere (12~. Briefly, after
being washed in Hanks' balanced salt solution (HBSS), cells were
incubated for 1 h with 1-2 ,uM fura 2-AM (in 10% Pluronic
F-127) at room temperature and under low light conditions.
Subsequently, fura 2-AM solution was removed and cells were
incubated for 1 h in HESS (50 ill) to allow endogenous esterases
to cleave AM ester. Cells were stimulated by addition of 50 al
of 2x tastant solution. Response kinetics were measured from
cells loaded with 100 ,u M fura 2 via patch pipette and stimulated
This paper results from the Arthur M. Sackier Colioquium of the National Academy of
Sciences, "Chemical Communication in a Post-Genomic Worid," held January ~ 7-19, 2003,
at the Arnold and Mabel Beckman Center of the National Academies of Science and
Engineering in Irvine, CA.
tTo whom correspondence should be addressed. E-mail: john.carlson~yale.edu. !
2003 by The National Academy of Sciences of the USA
www.pnas.org/cgi/doi/ 10.1 073/pnas.2 135339100
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Representative terms from entire chapter:
trehalose response
c
i
Fig. 1. Expression of GrSa in taste neurons in the labellum and distal
segments in the leg. Shown here are whole mount samples of labella or tars).
Genotypes examined were as follows: Gr5a-GAL4/+; UAS-lacZ/+ (a and c) and
Gr5a-GAL4/UAS-mCD8:GFP; GrSa-GAL4/UAS-mCD8:GfP(b and d9. Shown in b
is a composite of a series of confocal images. Reporter gene expression was
observed in #30 cells in each half of the labellum and 2 cells in each of the two
to three distal-most segments of the leg.
with lx test ant solution via a puffer pipette under control of
PicoPump 830 (World Precision Instruments, Sarasota, FL). All
chemicals were purchased from Sigma. Imaging experiments
were conducted on a Nikon TE300 inverted microscope
equipped with a Nikon superfluor lens ~ x 10/0.5 and x20/0.7s).
Cells were stimulated with 340 and 380 nm UP light from a
DeltaRAM monochromator, and resulting images were col-
lected by using an IC-200 intensified charge-coupled device
camera; data acquisition and analysis were performed by using
an ImageMaster suite (PTI, South Brunswick, NJ). Individual
responses were recorded for 60 s. F340/380 ratios were analyzed
to measure Ca2+ release. A responder cell was defined as one
showing a ratio of 0.34 or above. In general, >905to of responses
showed a ratio of at least 0.51.
Results
To test the hypothesis that GrSa plays a direct role in trehalose
reception, we first asked whether it is expressed in taste neurons.
Because previous attempts at in situ hybridization have proved
unsuccessful with the great majority of Or genes (3, 4) we
generated GrSa promoter-GAL4 lines. An 8.5-kb genomic re-
gion upstream of GrSa was used to supply a promoter, and GAL4
was used to drive expression of both UAS-lacZ and UAS-GEP
Chyb et a/.
0.8
o
._
co
o
o
o
.
.
-
o
.
.
.
.
60S
fig. 2. Time course of trehalose response in S2-GrSa cells. (Upper) A series of
images of a single fura 2-loaded S2-GrSa cell, taken at 5-s intervals. The first
image is taken 5 s before the application of 100 mM trehalose. (Lower) A
quantitative representation of the response of the same cell. Bar indicates
stimulus period.
reporters. We observed wide expression in taste neurons of the
labellum as well as in four to six neurons in the tarsi of adult flies
(Fig. 1~. No sexual dimorphism was observed in the expression
pattern. Six independently derived lines were examined and all
gave equivalent results.
To examine GrSa function at the cellular level, we expressed
GrSa cDNA in Drosophila S2 cells. This cell line was chosen for
two reasons. First, chemosensory receptors have been notori-
ously difficult to express in heterologous systems and we pre-
dicted that use of a Drosophila cell line might improve the
possibility of functional expression of a Drosophila receptor.
Second, previous studies have documented Ca2+ release after
activation of G protein-coupled receptors that couple to the
endogenous Gq protein of S2 cells (13-15~. In this system, ligand
binding to the receptor results in the activation of the phospho-
inositide (PI) pathway: hydrolysis of PIP2 by phospholipase C
into InsP3 and 4,5-diacylglycerol, and release of Ca2+ from
intracellular stores. The stimulus-activated change in [Ca2+]i can
be monitored with Ca2+-sensitive fluorescent ratiometric indi-
cators, such as fura 2 (16~.
We transiently expressed GrSa in S2 cells, loaded them with
100 ,uM fura 2, and applied 100 mM trehalose via puffer
pipette (Fig. 2~. Stimulation evoked Ca2+ release: cell response
developed within ~5 s of ligand application and reached a peak
intensity within ~15 s of application. Upon removal of the
ligand, the level of intracellular calcium gradually returned to
the baseline. These data provided initial evidence that GrSa
encodes a functional trehalose receptor when expressed in S2
cells. The results also suggested the possibility that GrSa-
encoded receptor protein couples to the endogenous phos-
phoinositide pathway. We then cotransfected S2 cells with
GrSa and promiscuous G proteins: G
pan me trehalose
25 AM trehalose
250 me maltose
250 EM trehalose
no GrSa receptor
2.5 mM trehalose
Fig. 3. Dose-dependence of trehalose response in S2-Gr5a cells. (Upper) Divided panels of 52-GrSa cells (Left and Center) or negative controls, transfected with
GFPvectoralone(Right), beforeandafterapplication of either250 mMtrehalose (Leftand Right) or250mM maltose (Center). (Lower) ImagesoffieldsofS2-Gr5a
cells taken on application of different concentrations of trehalose (indicated below).
,uM, and saturation was observed in the low mM range (Fig. 4a).
The Hill coefficient was nH = 1.92, suggesting the possibility that
GrSa functions as a homodimer.
We have investigated the ligand specificity of GrSa by
challenging S2-GrSa cells with other sugars. We first tested two
other dissacharides, maltose (composed of two glucose units)
and sucrose (one glucose unit linked to fructose), and found
that even when tested at high concentrations they elicited little
if any response, as measured in terms of the percentage of cells
that yielded a F340/380 - 0.34 (Figs. 3 and 4~. We then
systematically tested a number of common disaccharides struc-
turally related to trehalose, each composed of two glucose
units (Fig. 4b). These isomers varied only in the positions
of their glycosidic bond (e.g., 1,1', 1,4', or 1,6') and/or the
configuration (c' or ,`3) of the D-glucose subunits. None of these
other disaccharides evoked significant responses (Fig. 4c). The
isomers tested included isotrehalose and neotrehalose, which,
like trehalose, contain 1,1' linkages; however, isotrehalose
contains a I3,,B linkage, and neotrehalose contains an cz,,B
linkage, whereas trehalose contains an ~x,~ linkage. D-glucose,
a monosaccharide component of all of the disaccharides
tested, evoked no detectable Ca2+ release even at the highest
dose tested (250 mM). The simplest interpretation of these
results is that GrSa recognizes moieties close to the la,l'c~
glycosidic bond.
Trehalose is found in certain drought-adapted organisms such
as yeasts and is thought to play a role in protecting the membrane
during dehydration (19, 20~. Although trehalose has no effect on
Ca2+ levels in control cells that do not express GrSa, we carried
out two further experiments to control for the formal possibility
that trehalose interacts with the plasma membrane and somehow
activates the receptor nonspecifically. We found first that tre-
halose had no effect on cells transfected with another Gr gene,
Gr64f, and, second, that other molecules believed to have similar
effects on membranes, glycerol and 1,2 propanediol, have no
effect on GrSa-expressing cells (tested at 100 mM each, data not
shown). These results are consistent with the conclusion that
trehalose is a ligand for GrSa.
14528 1 www.pnas.org/cgi/doi/10. 1 073/pnas.21353391 00
D· ~
Iscusslon
This study provides direct evidence that GrSa, a member of a
large family of G protein-coupled receptors, functions as a
trehalose taste receptor. GrSa is expressed in neurons in taste
sensilla of both the labellum and tars), consistent with a role as
a taste receptor. When expressed in Drosophila S2 cells, it
confers a response to trehalose in a dose-dependent manner. The
response depends both on the expression of GrSa and on a
specific stimulus, trehalose. Other disaccharides tested evoke
little if any response in this system.
We have provided evidence that GrSa is expressed in all, or
almost all, of the ~33 sensilla present on the labellum. Because
the labellum responds to a variety of sugars, and because the
sensilla each contain a single sugar-sensitive neuron, the broad
expression we have observed is consistent with a model in which
many, if not all, of the sugar-sensitive taste neurons express more
than one receptor. This model is supported by our earlier finding
that mutation of GrSa affected the physiological response of the
sugar cell to trehalose, but not to sucrose, as if many of the
sugar-sensitive cells contain both a trehalose receptor, GrSa, and
a sucrose receptor (9~.
The expression pattern of GrSa is broader than that observed
for previously described GAL4 lines established by using pro-
moters of other Or genes (4, S). The broad pattern is consistent
with our earlier physiological data (9), which indicated that Gr5a
is required for trehalose response in all L- and M-type sensilla
(21~. Hiroi et al. (22) also found that most sensilla on the
labellum respond to trehalose.
We note that the response threshold of S2-GrSa cells to
trehalose is lower than in taste neurons in viva, as determined
in single-unit electrophysiological recordings (9). There are
several possible explanations for this difference. One is that
the two experiments measure different parameters, i.e., Ca2+
levels vs. action potential frequency, and the Ca2+ level we
have established as a criterion for scoring a response may be
less than that required to initiate action potentials. Another
possibility concerns access to tastant: in the expression system,
Chyb et a/.
a 1.25 1 C 60
1.00
a)
o
~ 0.7~
a)
N 0.50
o
0.2¢
0.00
/ &=~}~ Maltose
Sucrose
~ ~ Trehalose
0.025 0.25 2.5 25 250
[sugar] mM
b Trehalose Isotrehalose
OH
HO~—
H~_:
OH i
o
11
5~
-= 40-
o
Q 30-
tv 2
1O
~ _
HQ
~OH
b—~ OH
HO
OH
H~ H
~O~OH
OH O—:_ OH
HC)'
~ ~ 0 ~ (D 0 a) (D ~ 0
0 ~ u' cn cn oo ~ ~ 6o u'
o o o o o o o o o .
0 ~ 0 0 53 E ~ ~ C, Z
~ Z — ~
Neotrehalose
,OH
HO~—
HO~
OH -' ~
~O~ ~OH
Maltose Cellobiose Sucrose
OH
H~ j OH
~\
HO~OH
OH
OH OH OH
H~(Z~_OH
OH OH
Isomaltose Genbobiose Glucose
OH OH
H~ j H:Q:
HO:—
OH]
HO - ~
HO~_OH
OH
OH OH
OH
HO~—
H(~
OH]
HO~OH 1H
OH
Fig. 4. (a) Dos~response curves for selected disaccharides. Responses are normalized to response at 250 mM trehalose. 4 c n c 6; error bars = SEM. (b) Structures
of selected disaccharides related to trehalose. With the exception of sucrose, all are composed of 2 units of glucose. The cY-configuration is highlighted in yellow,
and the ,B-configuration of the C1 carbon is highlighted in red. Isotrehalose and neotrehalose do not occur naturally. (c) Specificity of trehalose response in
S2-GrSa cells. Shown here are the responses of GrSa-S2 cells to the various disaccharides illustrated in b. Compounds were tested at a concentration of 250 mM.
5 c n c 9. "No GrSa" cells were stimulated with 250 mM trehalose.
the cells are bathed in tastant, whereas in vivo the tastant must
enter a pore in a sensillum and diffuse into the lymph
surrounding a dendrite, where its final concentration may be
lower than that of the test solution. A third possible explana-
tion is that the density of receptor protein, or of another
signaling component, may be greater in the heterologous
expression system than in vivo.
The simplest interpretation of our results is that GrSa func-
tions as a homodimer, unlike the ~nammalian sweet receptors,
which function as heterodimers (12~. Furthermore, in contrast to
the TlR2/TlR3 mammalian receptor that is rather broadly
Chyb et a/
furled to diverse sweet-tasting molecules such as sucrose, sac-
charin, dulcin, and acesulfame-K, the GrSa receptor is tuned to
trehalose and shows much less, if any, response to other sugars,
such as sucrose, fructose, and glucose, which the fly encounters
in its natural habitat.
The relatively narrow tuning of GrSa has implications for the
mechanism of taste coding. If other Drosophila taste receptors
are as specific as GrSa, then an individual tastant is likely to be
encoded largely by the activity of one or a small number of
receptors, as opposed to the integrated activity of many recep-
tors, each exhibiting a varying degree of response to a ligand. In
PNAS I November25, 2003 1 vol. ~Oo 1 suppl. 2 1 14529
the olfactory system of Drosophila, many individual odorants
activate several distinct classes of receptor neurons (23, 24), each
expressing distinct odor receptors (ref. 25 and J.R.C., unpub-
lished results). This model of taste coding is also supported by
the severe loss of trehalose response after mutation of a single
receptor gene, GrSa. Analysis of further Or proteins will be
required to determine whether the narrow tuning of GrSa is
representative of Or receptors at large or of those that recognize
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