The Journal of Neuroscience, August 13, 2003, 23(19):7376-7380
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Functional Interaction between T2R Taste Receptors and G-Protein 
Subunits Expressed in Taste Receptor Cells
Takashi Ueda,1
Shinya Ugawa,1
Hisao Yamamura,1,2
Yuji Imaizumi,2 and
Shoichi Shimada1
1Department of Molecular Morphology, Graduate
School of Medical Sciences, Nagoya City University, and
2Department of Molecular and Cellular Pharmacology,
Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya
467-8601, Japan
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Abstract
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Bitter taste perception is a conserved chemical sense against the ingestion
of poisonous substances in mammals. A multigene family of G-protein-coupled
receptors, T2R (so-called TAS2R or TRB) receptors and a G-protein 
subunit (G), gustducin, are believed to be key molecules for its
perception, but little is known about the molecular basis for its interaction.
Here, we use a heterologous expression system to determine a specific domain
of gustducin necessary for T2R coupling. Two chimeric G16 proteins
harboring 37 and 44 gustducin-specific sequences at their C termini
(G16/gust37 and G16/gust44) responded to different T2R receptors with known
ligands, but G16/gust 23, G16/gust11, and G16/gust5 did not. The former two
chimeras contained a predicted
6 sheet, an5 helix, and an extreme
C terminus of gustducin, and all the domains were indispensable to the
expression of T2R activity. We also expressed G16 protein chimeras with the
corresponding domain from other Gi proteins, cone-transducin
(Gt2), Gi2, and Gz (G16/t2, G16/i2, and G16/z). As a
result, G16/t2 and G16/i2 produced specific responses of T2Rs, but G16/z did
not. Because Gt2 and Gi2 are expressed in the taste receptor
cells, these G-protein i subunits may also be involved in bitter taste
perception via T2R receptors. The present G16-based chimeras could be
useful tools to analyze the functions of many orphan G-protein-coupled taste
receptors.
Key words: bitter taste; T2R receptor; G-protein subunit; gustducin; G chimera; calcium imaging
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Introduction
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Taste perception is initially mediated by multiple signaling pathways in
the taste receptor cells (TRCs) within taste buds in the oral epithelium.
Bitter taste, as well as sweet taste, is believed to be detected by
G-protein-coupled receptors (GPCRs), and the signaling pathways of TRCs have
been the subject of intense speculation (for review, see
Gilbertson et al., 2000
;
Lindemann, 2001;
Margolskee, 2002;
Montmayeur and Matsunami,
2002). -Gustducin is a transducin-like Gi protein
selectively expressed in 20 -30% of TRCs
(McLaughlin et al., 1992).
In vitro biochemical assays and in vivo physiological
studies using knock-out mice have demonstrated that gustducin plays a key role
in TRC responses to numerous bitter compounds
(Wong et al., 1996). However,
gustducin knock-out mice still retained substantial sensitivity to bitter
compounds in physiological and behavioral assays
(Wong et al., 1996;
He et al., 2002). In contrast,
the second family of taste receptors identified, T2R (so-called TAS2R or TRB),
is a large GPCR multigene family of 
30 members in humans and rodents
(Adler et al., 2000;
Matsunami et al., 2000). The
genes map to regions of human and mouse chromosomes implicated genetically in
sensitivity to various bitter compounds and are coexpressed with gustducin,
suggesting that T2R receptors could function as gustducin-linked bitter taste
receptors. Currently, there are two T2R receptors that display ligand
responses with an affinity range compatible with behavioral sensitivity: mouse
T2R5 (mT2R5) for cycloheximide
(Chandrashekar et al., 2000)
and human T2R16 (hT2R16) for salicin (Bufe
et al., 2002). However, the other T2R receptors remain orphan
receptors with no known ligands.
To measure T2R activity, previous studies used a heterologous expression
system using human embryonic kidney 293/G15 (HEK293/G15) cells
(Chandrashekar et al., 2000;
Bufe et al., 2002). These cells
stably express the subunit of the mouse G-protein subunit
(G) protein G15, which is thought to indiscriminately couple to
many GPCRs (Offermanns and Simon,
1995). In this strategy, transfection of G15 into the cell
system potentially allows measurements of elevated levels of intracellular
calcium [Ca2+]i, giving a simple readout for agonist
activation, although there is evidence that bitter taste transduction is
mediated by Gi-coupled receptors. However, G15 cannot be
considered as a true universal adapter for GPCRs, because 18% of the
total number of Gi-coupled GPCRs examined to date cannot activate its
human ortholog G16 (Mody et al.,
2000; Kostenis,
2001). Moreover, T2R receptors are believed to couple with
gustducin in the native TRCs. In the present study, we constructed a variety
of chimeric G proteins to determine the specific domain of gustducin
necessary for T2R coupling and demonstrated that a specific domain of
gustducin is indispensable to ligand responses compatible with behavioral
sensitivity.
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Materials and Methods
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Materials. Animals were obtained from Shizuoka Laboratory Animal
Center (Shizuoka, Japan). The human leukemic cell line, HL60, was obtained
from the Japan Collection Research Bioresources Cell Bank (Japan). Reagents
for reverse transcription PCR (RT-PCR) were obtained from Invitrogen
(Carlsbad, CA) and Applied Biosystems (Branchburg, NJ). Cycloheximide was
purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Salicin,
serum, culture media, and anti-FLAG M2 and anti-opsin (Clone RET-P1)
monoclonal antibodies were from Sigma (St. Louis, MO) unless otherwise
noted.
Construction of G proteins and chimeras. A variety
of G subunits were obtained from a human cell line and rat tissues by
RT-PCR. Human G16 was obtained from HL60 cells. Rat -gustducin
and Gi2 cDNAs were from rat lingual tissues containing circumvallate
papillae. Similarly, rat Gt2 and Gz cDNAs were obtained from the
retina and brain, respectively. All of the chimeras were constructed by PCRs
using human G16 and rat-appropriated G cDNAs as templates. We
first constructed a series of G16/gustducin (G16/gust) chimeras by
incorporating different lengths of gustducin amino acid sequences at the C
terminus of G16: G16/gust44, G16/gust37, G16/gust23, G16/gust11, and
G16/gust5. In addition, we also constructed G16-based chimeras by
replacing 44 amino acid sequences at the C terminus of G16 with those
of Gt2, Gz, or Gi2 (G16/t2, G16/z, and G16/i2). All
full-length -subunit cDNAs were subcloned into a
pcDNA3.1(+) mammalian expression vector (Invitrogen). The
G16/gust chimeras were also tagged by a FLAG epitope and cloned into the
vector for expression assay and Western blot analysis.
Construction of T2R receptors. Mouse T2R5 and human T2R16 were
amplified from mouse and human genomic DNAs, respectively. We subcloned its
open reading frame into pME18S-FL3 containing the first 39 amino acids
of bovine rhodopsin in the frame. The sequences allow immunohistochemical
detection and facilitate expression of the recombinant chemosensory receptors
on the cell surface.
Transfection of HEK293T cells. HEK293T cells were cultured with
DMEM and supplemented with 10% FCS (v/v) at 37°C in humidified air with 5%
CO2. For transfection, cells were seeded onto 100 mm dishes or
uncoated glass coverslips in 35 mm chambers. After 24 hr at 37°C, cells
were washed in DMEM medium and transfected with G and T2R using
LipofectAmine 2000 reagent (Invitrogen). The transfection efficiencies were
estimated by cotransfection of a GFP reporter plasmid or by
immunohistochemistry and were typically >70%.
Western blot analysis. For Western blot analysis, a series of
FLAG-tagged G16/gust chimeras was used. Cells were grown on 100 mm dishes to
70-80% confluence. Transfection was performed on 35 mm dishes with proper
adjustments for the volumes and amounts of the reagents used. After 36 hr in
normal growth conditions, cells were spun down briefly, resuspended in a lysis
buffer (20 mM Tri-HCl, pH 7.4, 0.1% SDS, 1% Triton X-100, 1% sodium
decoxycholate) containing one protease inhibitor cocktail tablet (complete
mini; Roche Products, Mannheim, Germany), lysed by one cycle of freeze-thawing
followed by 10 passages through a 27-gauge needle at 4°C, and centrifuged
at 15,000 rpm for 30 min. Collected supernatants were used, and the protein
concentrations were determined using a Bio-Rad (Hercules, CA) protein assay
kit. Next, 50 µg of each protein sample was resolved on a 10%
SDS-polyacrylamide gel and transferred to Immobilon-P transfer membrane
(Millipore, Bedford, MA) via electroblotting. FLAG-tagged G chimeras
were detected by an anti-FLAG antibody followed by an alkaline
phosphatase-labeled anti-mouse IgG secondary antibody and then visualized by a
phosphatase reaction using nitro blue tetrazolium chloride and
5-bromo-4-chlor-indolyl-phosphate (Roche Products).
Calcium imaging. We used a cell-based reporter system to examine
T2R-G interaction (Chandrashekar et
al., 2000; Bufe et al.,
2002). In this system, receptor activation leads to increases in
intracellular calcium [Ca 2+]i, which can be monitored
at the single-cell level using the fura-2 AM calcium indicator dye. HEK293T
cells were transiently transfected with a rhodopsin-tagged T2R receptor with a
G16- or G16-based G chimera using LipofectAmine 2000
reagent. After 24 -30 hr, transfected cells were loaded with 5
µM fura-2 AM for 30 min at room temperature. The loading
solution was washed out, and cells were incubated in 500 µl of assay buffer
(10 mM HEPES, 130 mM NaCl, 10 mM glucose, 5
mM KCl, 2 mM CaCl2, and 1.2 mM
MgCl2, pH 7.4) and stimulated with tastants using a bath perfusion
system at a flow rate of 5 ml/min. We recorded [Ca 2+]i
changes using an Olympus IX-70 (Olympus Optical, Tokyo, Japan) equipped with
the ARGUS-HiSCA system (Hamamatsu, Shizouka, Japan). Aquisition and analysis
of the fluorescence images were done using ARGUS-HiSCA version 1.65 software.
Generally, [Ca 2+]i response was measured by
sequentially illuminating cells at 340 and 380 nm and monitoring the
fluorescence emission at 510 nm using a cooled CCD camera. At the beginning of
each experiment, 10 µM isoproterenol was applied for 10 sec. A
180 sec interval was then left between each tastant application to ensure that
cells were not desensitized as a result of the previous application of
tastants. In all cases, we measured the entire camera field. As a control, we
used isoproterenol (10 µM) to stimulate endogenous
-adrenergic receptors, proving that the G16-dependent signal
transduction cascade was functional. Approximately 70-80% of all cells in the
camera field responded to isoproterenol, whereas 15-20% of all cells in
the field showed dose-dependent responses to agonists in the transient
transfection experiments. The proportion of responders was about half of that
found by immunohistochemistry, which was similar to that in a previous study
using HEK/G15 cells (Bufe et al.,
2002).
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Results
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Figure 1A shows the
alignment of the C-terminal amino acid sequences of the G used. Murine
G15 has been successfully used to determine the function of two T2R
receptors when stably expressed in HEK293 cells. Because its human ortholog,
G16, is also known to interact with a wide variety of GPCRs, we first
examined whether G16 could couple to T2R receptors in our transient
expression system. Although we expressed G16 with mT2R5, a
cycloheximide receptor in the mouse, by transient transfection in HEK293T
cells, the T2R receptor did not respond to the cycloheximide
(Fig. 2). Similarly, hT2R16,
which has been reported to react with a plant bitter tastant salicin, failed
to respond to it (Fig. 2). In
both cases, 10 µM isoproterenol increased
[Ca2+]i by activating an endogenous -adrenergic
receptor present in HEK293T cells. Untreated cells and cells without
G16 did not respond to isoproterenol, indicating that human G16
could mediate intracellular calcium mobilization but could not couple to T2R
receptors in the present assay system. We thus used G16 as the basis of
G chimeras for a functional assay on the basis of calcium imaging.
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Figure 2. A, Schematic illustrations of chimeric G16/gust proteins with
different lengths of C terminal amino acids found in gustducin and their
abilities to couple to T2Rs. +, Specifically responded to the ligand in
dose-dependent manner; -, did not exhibit any responses. B,
Immunoblot analysis of FLAG-tagged chimeric G16/gust subunits expressed in
HEK293T cells, stained with the anti-FLAG M2 monoclonal antibody. The FLAG
epitope tag did not influence the functional activity of G16/gust
chimeras.
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As numerous studies on Gi subunits have attested to the importance
of the C-terminal tail of the subunit as one of the major receptor
contact regions, we constructed a series of G16/gustducin chimeras by
incorporating different lengths of gustducin sequences at the C terminus of
G16 (Fig. 1B).
First, because the 5 helix is a known contact region for receptors
(Lichtarge et al., 1996), we
replaced the entire 5 helix of G16 with that of gustducin. The
resultant chimera was named G16/gust23; for G16/gust23 and subsequent
chimeras, the number after the latter gust indicates the number of gustducin
residues present in the C terminus of the construct. Second, another region
determining coupling selectivity is the extreme C terminus (also called
C-terminal turn or -turn) (Conklin et
al., 1993; Blahos et al.,
2001). G16/gust5 contains the minimum sequences of gustducin that
correspond to the region decisive for coupling of G proteins with
specific receptors. G16/gust11 was designed from a G COOH-terminal
minigene vector to explore the coupling mechanisms of receptors
(Gilchrist et al., 2002).
Last, the region between 4 and 5 helices that includes the L9
loop and 6 sheet is also involved in coupling selectivity, probably by
directly interacting with the receptors of the rhodopsin-like family (family 1
GPCRs) (Noel et al., 1993).
G16/gust44 contains all the structures, an L9 loop, a 6 sheet, an
5 helix, and an extreme C terminus of gustducin, whereas G16/gust37
includes the latter three structures. We also made FLAG epitope-tagged
G16/gust chimeras, but the results obtained using these epitope-tagged
chimeras were identical to those using the G chimeras without epitope
tags.
We used a well established transient expression system to examine the
ability of the G16/gust chimeras to interact with T2R receptors (see Materials
and Methods). We first examined the response against cycloheximide in HEK293T
cells coexpressing the rhodopsin-mT2R5 (rho-mT2R5) with G16/gust44,
G16/gust37, G16/gust23, G16/gust11, or G16/gust5. When transfected with T2R
and either G16/gust44 or G16/gust37, cells specifically responded to
cycloheximide (Figs. 2 and
3). The response was receptor-
and G chimera-dependent, because cells lacking either of these
components did not cause a [Ca2+]i increase, even at a 1000-fold higher
cycloheximide concentration. In this assay, the EC50 value of mT2R5
was 0.5 µM, and the threshold was 0.2 µM
(Fig. 3). These responses
resembled those obtained from behavioral experiments with rodents (sensitivity
threshold, 0.25 µM), indicating that G16/gust44 and
G16/gust37 can functionally couple to mT2R5. In contrast, the other G-protein
chimeras (G16/gust23, G16/gust11, and G16/gust5) failed to mediate the
cycloheximide response under identical experimental conditions
(Fig. 2). These G16/gust
chimeras did not mediate any responses, even at 1000-fold higher ligand
concentration. Hence, we checked the expression of the G16 chimeras by Western
blot analysis using an anti-FLAG M2 monoclonal antibody for the
immunodetection. As shown Figure
2, all chimeras were detected by the antibody in proteins prepared
from HEK293T transfected with the FLAG-tagged chimeras. There were no
differences in protein expression between them
(Fig. 2).
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Figure 3. A, [Ca 2+]i responses in HEK293T cells
expressing G16/gust37 and rho-mT2R5 when treated with multiple pulses of 10
µM isoproterenol (iso), cycloheximide, and 3 mM PROP
(6-n-propylthiouracil). Isoproterenol was used to ascertain that the
G16-dependent signaling cascade was functional. Horizontal bars above the
traces indicate the time and duration of tastant pulses. Cycloheximide
triggered robust receptor activation, but PROP did not. Similar results were
obtained when G16/gust44 was used in place of G16/gust37. B,
Dose-dependent curves of the effects of the ligands on the [Ca
2+]i in cells expressing G16/gust37 and the T2R receptor
indicated.
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The ability of G16/gust44 and G16/gust37 to interact productively with the
mT2R5 receptor prompted us to further investigate their capacity to
functionally associate with another T2R receptor. HEK293T cells were
cotransfected with either G16/gust44 or G16/gust37 and rho-hT2R16. In
transfected cells, stimulation of the ligand for hT2R16 (salicin)
significantly increased [Ca2+]i
(Fig. 2). Such
[Ca2+]i increases were receptor- and G
chimera-dependent and were in a dose-dependent manner. The EC50 and
threshold were 2 and 0.2 mM, respectively
(Fig. 3). These closely
resembled those obtained in experiments with human subjects reported
previously (Bufe et al., 2002).
Thus, G16/gust44 and G16/gust37 successfully interacted with hT2R16 in
addition to mT2R5. However, the other chimeras (G16/gust23, G16/gust11, and
G16/gust5) did not mediate any ligand responses via hT2R16. Thus, the
C-terminal 37 amino acid residues containing the predicted 6 sheet,
5 helix, and extreme C terminus of gustducin may be necessary for
productive functional expression of T2R taste receptors.
It has been suggested that in native mammalian TRCs, bitter taste may also
be mediated by G proteins other than gustducin. We tested whether the
T2Rs studied could associate with the C-terminal domain of other G
proteins corresponding to the 6 sheet, 5 helix, and extreme C
terminus of gustducin. We constructed G16 chimeras with 44 amino acid C
termini found in a variety of Gi proteins, including Gt2,
Gi2, and Gz (Fig.
1A) (G16/t2, G16/i2 and G16/z), and assayed them under
identical experimental conditions to the experiments of G16/gust chimeras. As
a result, G16/t2 and G16/i2 exhibited effective couplings with both mT2R5 and
hT2R16 whereas G16/z did not (Fig.
4A). The dose-dependent curves were similar to those
obtained from G16/gust chimeras that coupled to these receptors
(Fig. 4B). There are
no significant differences in the potency and efficacy between G16/t2 and
G16/i2.
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Figure 4. A, Schematic illustrations of chimeric G16 proteins with 44 C
terminal amino acids of different G proteins and their ability to
couple to T2Rs. +, Specifically responded to the ligand in a dose-dependent
manner; -, did not exhibit any responses. B, Dose-dependent curves of
the effects of ligands on the [Ca 2+]i in cells
expressing G16/t2 or G16/i2 and the T2R receptor indicated.
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Discussion
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To determine essential domains for coupling to GPCR, many
loss-of-function-type mutation studies on G-proteins have been done. However,
mutations of structurally important G-protein domains can also inhibit
functional coupling with GPCRs, although the mutation domains themselves are
not binding sites for GPCRs. In the present study, we constructed a variety of
chimeric G proteins to better understand the domains involved in the
interactions between T2R and gustducin. In addition, these chimeric G
proteins enabled us to analyze gustducin-linked GPCRs on common robust assays
that are amenable to high throughput-screening analysis. We monitored
[Ca2+]i increases caused by activation of signaling
cascade T2R-heterotrimeric G-protein (G
)-phospholipase
C-inositol 3,4,5 triphosphate receptor. From the G-protein , ,
and subunits, probably both G and dimers contact
the receptors. The G subunit is likely to play a decisive role in
discriminating between different receptor subtypes
(Savarese and Fraser, 1992;
Bourne, 1997;
Wess, 1997). Here, we
demonstrated that G16/gust44 and G16/gust37 successfully coupled to T2R
receptors for their signal transduction. These responses were comparable with
those obtained from in vivo experiments, and there is evidence that
mT2R5 can activate intact gustducin in vitro
(Chandrashekar et al., 2000),
indicating that our system is capable of reproducing signaling transduction of
T2R receptors in native TRCs. In contrast, G16/gust23 that contained the
5 helix of gustducin appeared not to associate, although numerous
studies have attested to the importance of the 5 helix in receptor
coupling. Similarly, G16/gust11 and G16/gust5 did not cause T2R activity.
These results indicated that the 5 helix and extreme C terminus of
gustducin were insufficient for detection of T2R activities, and the 6
sheet, in addition to the 5 and C-terminal -sheet, is
indispensable for signal transduction of T2Rs.
We next tested whether T2Rs could couple to domains including the 6
sheet, 5, and extreme C terminus from other G-protein subunits.
As a result, we revealed that some G16 chimeras constructed from other
G proteins, G16/t2 and G16/i2, functionally coupled with the T2R
receptors examined. Rod--transducin (Gt1) and
cone--transducin (Gt2) are present in vertebrate taste cells.
The former has been reported to transduce bitter taste by coupling taste
receptor(s) to taste cell phosphodiesterase
(Ruiz-Avila et al., 1995). In
addition, gustducin and rod-transducin are biochemically indistinguishable in
their in vitro interactions with retinal phosphodiesterase, rhodopsin
(retinal GPCR), and G-portion subunits
(Hoon et al., 1995). Because
the amino acid sequences of the 6 sheet and 5 helix in Gt1
and Gt2 are almost identical to those of gustducin
(Fig. 1A), the
C-terminal region (6, 5, and extreme C terminus) conserved could
be one of the most important domains for -gustducin, Gt1, and
Gt2 to interact with GPCRs.
In the present study, functional expression of T2R receptors was also
observed in HEK293T cells coexpressing G16/i2 and T2R receptors, suggesting
that T2Rs cannot only couple to gustducin and transducin but also to the
G-protein i2 subunit. One or more G-protein subunits may play a
role in bitter taste transduction, because -gustducin knock-out mice
retain residual responsiveness to bitter compound. In addition, transgenic
expression of a dominant-negative form of -gustducin from the gustducin
promoter further decreased the residual responses of -gustducin
knock-out mice apparently by inhibiting T2R/TRB interactions with other
TRC-expressed G-protein subunits
(Margolskee, 2002). It has
been reported that Gi2 subunit is expressed in subsets of TRCs. The
frequency of Gi2 expression appears to be higher than that of
gustducin, and some Gi2-positive cells also express -gustducin
(Kusakabe et al., 2000).
Gi2 could thus function as "backup" for gustducin in
T2R-gustducin-expressing TRCs. In contrast, several TRCs that are
immunoreactive for Gi2 but not for gustducin responded to cycloheximide
in an in vivo recording using mouse lingual slices
(Caicedo et al., 2002). This
suggests that Gi2 may be involved in gustducin-independent bitter taste
transduction via other G-protein-coupled receptors as well as T2Rs. On the
basis of in situ hybridization with a mix of 10 different T2R probes,
it was concluded that T2R genes are selectively expressed in
gustducin-expressing taste receptor cells
(Adler et al., 2000). However,
a recent genomic study has shown that mouse T2R (Tas2r) family is composed of
at least 36 full-length genes (Shi et al.,
2003). Additional studies are required to determine whether all of
the T2R genes are exclusively expressed in gustducin-expressing cells.
Many Gi-coupled GPCRs share the ability to inhibit adenylyl cyclase
via the pertussis toxin-insensitive Gz
(Chan et al., 1995;
Lai et al., 1995). The
incorporation of a Gz-specific sequence into a G16 backbone
(G16/z) successfully improved the recognition of a variety of
Gi-coupled receptors (Mody et al.,
2000). However, the G16/z chimera was incapable of responding to
the T2Rs studied in the present experiments, indicating that T2R receptors
have specific sequences for interacting with gustducin and Gi2. Within
the 6 sheet and 5 helix (37 amino acids), there are five amino
acids that are conserved in gustducin and Gi2 but not in Gz:
V333, K346, D350, C351, and F354 in gustducin (at position -23, -10, -5, -4,
and -1, respectively; the residues -1 being the last one)
(Fig. 1A). In
particular, the latter three amino acids are contained in the extreme C
terminus of the G protein. Indeed, a gustducin mutant containing a
glycine-to-proline substitution at position -3 can bind to taste receptor
G subunits and the effector, but it cannot be activated by
receptors (Ruiz-Avila et al.,
2001). Therefore, the extreme C terminus may also play an
important role in transduction via the gustducin, Gt1, Gt2, and
Gi2 of T2R taste receptors.
In conclusion, we found that 37 C terminal amino acids (6, 5,
and extreme C terminus) of gustducin and Gi2 are indispensable for the
detection of T2R activity. Because T2Rs have the greatest conservation in
their cytoplasmic loops and adjacent transmembrane segments, which are the
predicted sites for G-protein interaction, the present chimeric G16/gust
proteins could be powerful tools to analyze orphan gustducin-linked taste
receptors.
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Footnotes
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Received May. 6, 2003;
revised Jun. 9, 2003;
accepted Jun. 18, 2003.
This work was supported by research grants from the Japan Society for the
Promotion of Science.
Correspondence should be addressed to Takashi Ueda, Department of Molecular
Morphology, Graduate School of Medical Sciences, Nagoya City University, 1
Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail:
tueda{at}med.nagoya-cu.ac.jp.
Copyright © 2003 Society for Neuroscience
0270-6474/03/237376-05$15.00/0
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Abstract
Introduction
