The G Protein-Coupled Taste Receptor T1R1/T1R3
Regulates mTORC1 and Autophagy
Eric M. Wauson,1Elma Zaganjor,1A-Young Lee,1Marcy L. Guerra,1Anwesha B. Ghosh,1Angie L. Bookout,1
Chris P. Chambers,2Arif Jivan,1,3Kathleen McGlynn,1Michele R. Hutchison,2Ralph J. Deberardinis,2
and Melanie H. Cobb1,*
1Department of Pharmacology
2Department of Pediatrics
University of Texas Southwestern Medical Center, Dallas, 6001 Forest Park Road, Dallas, TX 75390-9041, USA
3Present address: Northwestern University Feinberg School of Medicine, Arthur J. Rubloff Building, 420 East Superior Street,
Chicago, IL 60611, USA
Cells continually assess their energy and nutrient
state to maintain growth and survival and engage
necessary homeostatic mechanisms. Cell-autono-
mous responses to the fed state require the surveil-
lance of the availability of amino acids and other
nutrients. The mammalian target of rapamycin com-
plex 1 (mTORC1) integrates information on nutrient
and amino acid availability to support protein syn-
thesis and cell growth. We identify the G protein-
coupled receptor (GPCR) T1R1/T1R3 as a direct
sensor of the fed state and amino acid availability.
Knocking down this receptor, which is found in
most tissues, reduces the ability of amino acids to
signal to mTORC1. Interfering with this receptor
alters localization of mTORC1, downregulates ex-
pression of pathway inhibitors, upregulates key
amino acid transporters, blocks translation initiation,
and induces autophagy. These findings reveal a
mechanism for communicating amino acid avail-
ability through a GPCR to mTORC1 in mammals.
The mammalian target of rapamycin (mTOR) coordinates cell
growth, protein translation, and autophagy with the availability
of nutrients, growth factors, and energy (Dazert and Hall, 2011;
Avruch et al., 2009; Ma and Blenis, 2009; Zoncu et al., 2011;
Corradetti and Guan, 2006). At least two functionally distinct
mTOR complexes (mTORCs) have been identified, mTORC1
and mTORC2, that carry out different cellular functions.
mTORC1 is regulated by insulin and growth factors by mech-
anisms primarily dependent on phosphatidylinositol 3-kinase
(PI3K) and Ras. These pathways relieve mTORC1 repression
by tuberous sclerosis 1 and 2 (TSC1/TSC2). TSC1/TSC2 are
GTPase-activating proteins that inactivate RAS homolog en-
riched in brain (Rheb), which is required for the activation of
mTORC1. Akt and ERK1/2, through the ERK1/2 substrate ribo-
somal protein S6 kinase (RSK), phosphorylate and inhibit the
TSC1/TSC2 complex (Roux et al., 2004; Carrie `re et al., 2008),
releasing its negative control of mTOR and enhancing mTORC1
activity. ERK1/2 may also promote mTORC1 activation by
directly phosphorylating Raptor, a subunit of mTORC1 (Carrie `re
et al., 2008; Carriere et al., 2011).
Among its most important functions, mTORC1 stimulates
protein translation. Cap-dependent translation initiation involves
capped 50end of mRNA, and its scanning to the start codon to
form a complex with the large ribosomal subunit. In the transla-
tionally inactive state, the 7-methylGTP cap binds eIF4E and its
inhibitor eIF4E binding protein 1 (4EBP-1) (Ma and Blenis, 2009;
Sonenberg and Hinnebusch, 2009). Amino acid repletion and
growth factors induce phosphorylation of 4EBP-1, releasing it
from eIF4E and enhancing recruitment of eIF4G, other initiation
factors, and the 40S ribosomal subunit to the mRNA cap.
mTORC2 localizes to ribosomes to phosphorylate Akt and
some of its relatives on hydrophobic motif sites required for
high activity (Oh et al., 2010).
Nutrient starvation suppresses mTORC1 more than mTORC2,
leadingtoincreasedratesof catabolic processesincludingauto-
phagy (Dazert and Hall, 2011; Avruch et al., 2009; Zoncu et al.,
2011; Ma and Blenis, 2009; Corradetti and Guan, 2006).
Damaged organelles and long-lived proteins may be trapped in
autophagosomes, which then fuse with lysosomes to degrade
their contents. Under stress, autophagy can accelerate to pro-
vide nutrients for enhanced survival. With sufficient amino acids,
mTORC1 phosphorylates and inhibits the autophagy kinase
unc51-like protein kinase ULK1 slowing autophagy (Jung et al.,
2009; Egan et al., 2011).
Several events have been implicated in mTORC1 activation
by amino acids. The Rag GTPases associate with mTORC1 in
an amino acid-dependent manner and localize it to a compart-
ment that contains its activator Rheb. Also involved is the Ragu-
lator complex that scaffolds mTORC1 to late endosomes and
lysosomes, where it is activated. The spatial organization of
lysosomes in the cytoplasm is critical for mTORC1 activation
(Sancak et al., 2010).
Preceding these key steps, the initial amino acid-sensing
mechanisms are less clear. Amino acids are transported into
cells where it is thought that they engage an unknown
Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc. 851
porters themselves are proposed to be components of the
sensory apparatus. The bidirectional amino acid transporter
SLC7A5/SLC3A2 is thought to activate mTOR by allowing for
the influx of leucine in exchange for the efflux of glutamine
(Nicklin et al., 2009). Yeast Ssy1, a member of the amino acid
permease family, regulates gene transcription in response to
extracellular amino acids, although it is not able to transport
amino acids into the cell (Hundal and Taylor, 2009; Forsberg
and Ljungdahl, 2001). Glutamatergic activation of mTORC1
has also been reported in neurons by Lenz and Avruch (2005).
Findings such as these suggest possible roles for membrane
receptors in signaling to mTORC1.
The G protein-coupled receptor (GPCR) complex T1R1/T1R3
is an amino acid receptor that was discovered in gustatory
neurons as a detector of the umami flavor (Matsunami et al.,
2000; Nelson et al., 2002). The related receptor T1R2/T1R3,
also discovered in gustatory neurons, is known as the sweet
receptor and functions as a glucose sensor not only in gustatory
neurons but also in the intestine and the hypothalamus (Ren
et al., 2009; Jang et al., 2007; Mace et al., 2007; Nakagawa
et al., 2009). Here, we show that T1R1/T1R3 is an early sensor
of amino acid availability. Reduced expression of this recep-
tor impairs activation of mTORC1 by amino acids, results in
mTORC1 mislocalization, and accelerates autophagy.
The GPCR T1R1/T1R3 Is an Amino Acid Sensor
in Pancreatic b Cells
Pancreatic b cells respond to increased nutrient concentrations
by secreting insulin and inducing synthetic processes to
replenish the insulin supply. Activation of ERK1/2 by glucose in
b cells is necessary for glucose-stimulated insulin gene tran-
scription (Khoo et al., 2003); yet, the mechanism(s) by which
these cells sense and respond to amino acids is unclear.
Because ERK1/2 are activated by glucose and hormones that
may alsomonitorextracellular amino acids.Wenoted thatamino
acids rapidly yet transiently activated ERK1/2 in MIN6 b cells. In
b cells the kinetics of ERK1/2 activation by amino acids and by
carbachol, a ligand for muscarinic (M3) GPCRs, were similar
if other aspects of the mechanism were similar. Muscarinic
GPCR activation increases phospholipase C b (PLCb) activity,
which leads to an influx of extracellular calcium. We found that
amino acids induced calcium entry into MIN6 cells with similar
kinetics and amplitude as carbachol, and calcium entry was
necessary for ERK1/2 activation by both ligands (Figures 1B
and 1C). PLCb activation was required for amino acid-induced
calcium entry and ERK1/2 activation because the PLCb inhibitor
U73122 blocked both of these events (Figure 1D; see Figure S1A
available online), revealing common mechanistic features.
These data suggested that amino acids stimulated ERK1/2
through a cell surface GPCR. Because the GPCR complex
T1R1/T1R3 is activated by all 20 amino acids, we tested the
hypothesis that T1R1/T1R3 was the amino acid sensor in
pancreatic b cells signaling to ERK1/2. We observed that amino
ing either T1R3 or T1R1 shRNA was significantly reduced com-
pared to cells expressing a control shRNA (Figures 1E–1H and
S1E). Carbachol-induced ERK1/2 activation was not diminished
with T1R3 knockdown, demonstrating that reduced expression
of T1R3 leaves other signaling mechanisms to ERK1/2 intact
Expression of T1R1/T1R3 was initially thought to be re-
stricted to gustatory neurons (Matsunami et al., 2000; Nelson
et al., 2002). Because most cell types require amino acid-
sensing capabilities to coordinate cellular demands with
nutrient availability, we asked if T1R1/T1R3 might serve as an
amino acid sensor in other cell types as well. We found that
both T1R1 and T1R3 are present broadly in mouse tissues,
human islets, and many types of cultured cells (Figures 1I and
Amino Acids Signal to mTORC1 through T1R1/T1R3
mTORC1 coordinates cell growth with the availability of amino
acids. That mTORC1 is activated by amino acids is well charac-
terized, but less is understood about upstream mechanisms. In
MIN6 cells in which either T1R3 or T1R1 was knocked down,
there was a significant impairment of amino acid-induced phos-
phorylation of the mTORC1 substrate p70 ribosomal protein S6
kinase (S6K) (Figures 2A and S2A). The phosphorylation of
mTOR on S2488, which is positively correlated with increased
mTORC1 activity (Nave ´ et al., 1999), was also reduced in the
T1R3 and T1R1 knockdown cells (Figure S2B).
Because we observed robust T1R1 and T1R3 expression in
mouse heart (Figure S1D), we investigated the role of T1R1/
T1R3 signaling to mTORC1 in H9C2 rat cardiac myoblasts. We
depleted T1R1 and T1R3 transiently in H9C2 cells using siRNA
and found that amino acid-stimulated and basal mTORC1
activity, assessed by phosphorylation of both S6K on S389
and ribosomal protein S6 on S235 and S236, was reduced
(Figures 2B–2D, S2D, and S2G).
Activation of mTORC1 by growth factors requires the pres-
ence of amino acids (Avruch et al., 2009). Because we hypothe-
sized that T1R1/T1R3 provides this amino acid signal, we
determined if knockdown of this receptor would inhibit growth
factor-induced mTORC1 activation. We observed that reduced
T1R1 expression in H9C2 cells significantly reduced the ability
of EGF and fetal bovine serum (FBS) to induce phosphorylation
of S6K and S6 even in the presence of amino acids (Figures
2B, 2C, S2D, and S2G). As additional support for the conclusion
that T1R1/T1R3 is an amino acid sensor for mTORC1 activation,
we acutely impaired human T1R3 signaling using small molecule
inhibitors (Xu et al., 2004; Maillet et al., 2009). Pretreatment of
cells with lactisole for 15 min reduced amino acid-stimulated
phosphorylation of S6K and S6 in HeLa cells in a dose-depen-
dent manner (Figures 2E, 2F, and S2E). Amino acid-induced
phosphorylation of S6K in PANC-1, MCF7, and Jurkat human
cell lines was also reduced by receptor inhibitors (Figure S2F).
Thus, T1R1/T1R3 senses amino acids and communicates this
information to mTORC1 in a wide variety of human and rodent
Amino acids activate mTORC1 in part by localizing the kinase
to lysosomal membranes where it interacts with Rag small
T1R1/T1R3 Regulates mTORC1
852 Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc.
GTPases and the Ragulator complex (Sancak et al., 2010). We
tested the idea that T1R1/T1R3 may be necessary to trigger
the movement of mTORC1 to lysosomes. In control cells we
observed an amino acid-induced redistribution of mTOR from
a diffuse cytoplasmic pattern to a more concentrated localiza-
tion, in proximity to the lysosomal marker LAMP2 (Figure 2G).
However, amino acids did not cause redistribution of mTOR in
cells with reduced T1R3 expression, nor did mTOR appear to
colocalize to a substantial extent with LAMP2 in those cells.
LAMP2 expression was greater, whereas S6 phosphorylation
was decreased in the receptor knockdown cells (Figures 2H
and 2I). The distribution of this lysosomal membrane protein
also reflected a reorganization of lysosomes; they appeared
more diffuse and peripheral in the cytoplasm, a pattern charac-
teristic of starved cells. These results suggest that T1R1/T1R3
Figure 1. Amino Acids Activate ERK1/2
through the T1R1/T1R3 Receptor
(A) MIN6 pancreatic b cells in Krebs-Ringer
bicarbonate solution (KRBH) without amino acids
(aa) for 2 hr were treated with aa as defined in
carbachol (Carb) for the indicated times. ERK1/2
activation was analyzed by immunoblotting.
(B) MIN6 cells were treated as in (A) loaded with
fura-2 and then stimulated with aa or Carb as
above. Data are the mean ± SEM of the 340/380
values of three independent experiments.
(C) MIN6 cells incubated in KRBH for 2 hr as
above. Five minutes prior to stimulation with aa,
KCl, or Carb cells were placed in KRBH with or
without CaCl2. Cells were stimulated for 2 min,
harvested, and lysates were immunoblotted as
(D) Calcium was measured as above in MIN6 cells
pretreated with vehicle (DMSO), 10 mM U73122, or
10 mM U73344 for 30 min prior to aa stimulation.
Data are the mean ± SEM of the 340/380 values of
three independent experiments.
(E–H) MIN6 cells stably expressing control (Con)
shRNA1 and T1R3 shRNA1 (E and G) or T1R1
or 100 mM Carb for 2 min (E) or with aa for the
indicated times (F).
(I) RNA was isolated from tissues from C57BL/6J
mice for qPCR analysis. Data are normalized to
18S RNA and are the means of triplicate
measurements ±SD. Data from all panels are
representative of three independent experiments
except (E), one of eight, and (F), one of two. See
also Figure S1.
is involved in early events in amino acid
sensing that lead to mTORC1 activation.
Loss of T1R1/T1R3 Reduces
Translation Initiation and Insulin
Content of b Cells
mTORC1 controls translation initiation
(Ma and Blenis, 2009). In starved cells
4EBP-1 binds eIF4E, blocking initiation
of translation from capped mRNAs. Phosphorylation of 4EBP-1
by mTORC1 frees eIF4E to recruit eIF4G, the small ribosomal
subunit, and other factors to these mRNAs to initiate their trans-
lation (Brunn et al., 1997). Stable knockdown of T1R3 in MIN6
cells or transient knockdown of T1R1 in H9C2 cells reduced
phosphorylation of 4EBP-1 (Figures 3A, S3D, and S3E). Amino
acids were much less effective in recruiting eIF4G to the
to control (Figures 3B, 3C, and S3A–S3C). We conclude that
amino acid sensing through T1R1/T1R3 supports enhanced
cap-dependent translational activity.
ulated by insulin demand and is dependent on mTORC1 activity
(Welsh etal.,1986;Morietal., 2009). Basaland amino acid-stim-
ulated insulin secretion was reduced in T1R3 knockdown cells
T1R1/T1R3 Regulates mTORC1
Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc. 853
(Figure 3D). To determine the basis for decreased secretion, we
examined insulin protein content in control and T1R3 knock-
down MIN6 cells (Figure 3E). Immunoreactive insulin was
decreased by approximately 50% relative to control cells. A
smaller but significant loss was also noted in T1R1 knockdown
cells. Quantitation of preproinsulin mRNA indicated no reduction
in message (Figure 3F), suggesting that loss of insulin protein is
not due to loss of mRNA but rather due to insulin degradation
or insufficient translation or processing resulting from reduced
Activation of mTORC1 Is Reduced in T1R3–/–Mice
To determine the impact of T1R3 deficiency in mice, we
measured mTORC1 activity in the skeletal muscle and heart
- + + + +
-- + +
siRNA Control T1R3
aa- - - + + + - - - + + +
siRNA Control T1R1
aa - +
FBS - -
- + - +
+ + -
9 2746 442326 30
aa--+ + -- + +
1243 41 12108
aa -- + +-- + +
24440.1 0.1 0.1 0.2
1145 0.1 0.122
siRNA Control T1R3
siRNA Control T1R3
aa- + -
-- + - + -
--- + - + - -
- + + - + --+ +
- - + -
10 33 18 23 54 39171655320
Figure 2. The T1R1/T1R3 Taste Receptor Is
Necessary for Optimalaa-InducedmTORC1
Activation in Numerous Cell Types
(A) MIN6 cells stably expressing either control
shRNA2 or T1R3 shRNA1 in KRBH for 2 hr were
treated with aa or vehicle (H2O) for 10 min. Protein
expression and phosphorylation were analyzed by
immunoblotting. Signals were quantitated with the
under lanes are arbitrary units of pS6K/S6K. The
means of pS6K/S6K ± SEM from three indepen-
dent experiments are the following: Control (2 ±
0.42), Control + aa (33 ± 4.2), T1R3 (2 ± 0.44), and
T1R3 + aa (8 ± 0.98). The p values for Control ? aa
versus Control + aa, T1R3 versus T1R3 + aa, and
Control + aa versus T1R3 + aa are all <0.01.
(B) H9C2 cardiac myoblast cells transiently trans-
fected with either control nontarget siRNA1 or
T1R1 siRNA1 in KRBH were treated with vehicle,
aa, 100 nM insulin, or 50 ng/ml EGF for 30 min.
Protein expression and phosphorylation were
from pS6 relative to S6 or a-tubulin ± SEM from
three samples from two independent experiments
are Control (12.5 ± 3.3), Control + aa (33.9 ± 3.9),
Control + insulin (19.3 ± 1.0), Control + EGF (26.9 ±
3.2), Control + insulin and aa (43.2 ± 6.8), Control +
EGF andaa(43.9 ±3.7)T1R1(1.9 ±0.3),T1R1+aa
(8.3 ± 1.6), T1R1 + insulin (19.5 ± 2.5), T1R1 + EGF
(6.7 ± 1.5), T1R1 + insulin and aa (48 ± 6.0), and
T1R1 + EGF and aa (22.8 ± 4.6). The p values are
the following: Control + aa versus T1R1 + aa
Control + EGF versus T1R1 + EGF (<0.01),
Control + insulin and aa versus T1R1 + insulin and
aa (ns), and Control + EGF and aa versus T1R1 +
EGF and aa (<0.05).
(C) H9C2 cells transfected with the indicated
siRNAs as in (B) were stimulated with aa or 20%
FBS for 30 min. Quantitation was performed as
above. The means of pS6/S6 ± SEM for three
independent experiments are Control (10.3 ± 1.3),
Control + aa (30.7 ± 1.9), Control + FBS (67.3 ±
10.7), T1R1 (2.3 ± 0.3), T1R1 + aa (8.7 ± 2.9), and
T1R1 + FBS (36.7 ± 5.8). The p values are the
following: Control + aa versus T1R1 + aa (<0.01)
and Control + FBS versus T1R1 + FBS (<0.05).
(D) H9C2 cardiac myoblasts transiently trans-
fected with control siRNA1 or T1R3 siRNA1 in
KRBH were treated with vehicle or aa for 30 min. Immunoblots were performed as above. The means from three independent experiments ±SEM are the
following: control siRNA (1.7 ± 0.2), control siRNA + aa (5.4 ± 0.7), T1R3 siRNA (0.5 ± 0.2), and T1R3 siRNA + aa (2 ± 0.2). The p values of control siRNA versus
T1R3 siRNA and control siRNA + aa versus T1R3 siRNA + aa are 0.019 and 0.018, respectively.
(E and F) HeLa cells in KRBH for 2 hr were pretreated with 8 mM lactisole or vehicle (DMS0) for 15 min, and stimulated with aa for 30 min. (F) Graphed are mean ±
SEM of the ratio of pS6K/S6K from three independent experiments.
(G–I) HeLa cells transiently transfected with either scrambled siRNA or T1R3 siRNA4 were incubated in Earle’s Balanced Salt Solution (EBSS) for 90 min before
stimulation with aa for 30 min. Cells were then either fixed and stained with DAPI (blue), anti-mTOR (green), or anti-LAMP2 (red) antibodies (G) or lysed and
immunoblotted as above (H and I). (I) Means of three independent experiments ±SEM. **p < 0.01 and ***p < 0.001, two-tailed Student’s t test. See also Figure S2.
T1R1/T1R3 Regulates mTORC1
854 Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc.
from wild-type and T1R3 knockout mice fasted overnight. There
was an ?25% reduction in S2448 phosphorylation of mTOR in
the skeletal muscle and a 60% reduction in S6 phosphorylation
in hearts of T1R3 knockout mice compared to wild-type mice
(Figures 4A–4C and S4).
Because serum amino acid concentrations fluctuate as a
consequence of food intake, we asked if mTORC1 activity was
lower in the T1R3 knockout mice because of an overall decrease
in serum amino acid concentrations. We found that most amino
acid concentrations were unchanged between control and T1R3
knockout mice. Exceptions include a slight, but statistically
significant, decrease in asparagine and increase in glutamate
in the T1R3 knockout mice (Figure 4D).
A previous study reported that the concentrations of a number
of amino acids in plasma, skeletal muscle, and liver decreased in
C57BL/6J mice after 6–12 hr of fasting, with a transient increase
after 24 hr of fasting (Ezaki et al., 2011). Because agonist avail-
ability can induce compensatory changes in the expression of
receptors, we tested if T1R3 receptor expression was affected
in the skeletal muscle of C57BL/6J mice that were fasted for 6
and 12 hr. Immunoreactive T1R3 in skeletal muscle was highest
following a 12 hr fast (Figures 4E and 4F), and receptor mRNA
was also increased 3.5-fold (Figure 4G). Similar changes were
also observed in cultured cells (data not shown). As expected,
the phosphorylation of both AKT and S6 was reduced in the
muscle of mice after 6 and 12 hr of fasting when compared to
Signaling Mechanisms that Contribute to mTORC1
Amino acid regulation of mTORC1 may occur in part through
ERK1/2 directly and through the ERK1/2 substrate kinase RSK
(Roux et al., 2004; Carrie `re et al., 2008). Although it has been re-
ported that ERK1/2 are not sensitive to amino acids (Gulati et al.,
2008), we found that amino acids rapidly but transiently stimu-
lated ERK1/2 activity at concentrations as little as one-quarter
of those in growth medium, comparable to the sensitivity of
mTORC1 to amino acids (Figure 5A). Because we observed
thatT1R1/T1R3 signalsto ERK1/2(Figures 1Eand 1F),wedeter-
mined if ERK1/2 contribute to mTORC1 activation by amino
acids. U0126, which prevents ERK1/2 activation by blocking
MEK1/2, inhibited not only amino acid ERK1/2 stimulation but
also phosphorylation of S6K, implicating ERK1/2 in mTORC1
regulation in response to amino acids in multiple cells types
(Figures 5B, 5C, S5A, and S5B). These results suggest that
mTORC1. In contrast, AKT, another activator of mTORC1, was
not activated to a detectable extent by amino acids. Depletion
of T1R1 had little if any effect on serum stimulation of Akt
In gustatory neurons, amino acid binding to T1R1/T1R3
activates the G protein gustducin to stimulate PLCb2, which
leads to a release of calcium from intracellular stores. The role
of PLCb activity in mTORC1 activation was tested by comparing
the effects of the PLCb inhibitor U73122 to its inactive analog
U73343. The PLCb inhibitor, but not U73343, inhibited calcium
influx induced by amino acids (Figure 1D), and reduced amino
acid-induced mTORC1 activity (Figure 5E), suggesting that
Figure 3. Signaling through T1R1/T1R3 Is Necessary for Translation
as in Figures 1 and 2. Cells were treated with or without aa for 30 min.
Representative immunoblots of total 4EBP/1 and p4EBP from three inde-
pendent experiments are displayed.
(B) To investigate the ability of cells to initiate translation with T1R3 silenced,
Sepharose beads conjugated to 7-methylGTP. Proteins in lysates and bound
to 7-methylGTP were detected by immunoblotting.
(C) Amounts of eIF4G and eIF4E in lysates and bound to the 7-methylGTP
beads in (B) were quantified using the Licor Odyssey imaging system and
graphed as the ratio of bound eIF4G to total eIF4G in the lysate. Data are
mean ± SEM of four samples.
(D and E) Insulin was measured in the medium (D) or lysates (E) of stable MIN6
cell lines by ELISA and normalized to soluble protein. Mean ± SEM of three
samples from two independent experiments (D). Insulin measurements in (E)
are mean ± SEM of four independent experiments.
actin RNA and are mean ± SEM of triplicate measurements. *p < 0.05, **p <
0.01, and ***p < 0.001, two-tailed Student’s t test. See also Figure S3.
T1R1/T1R3 Regulates mTORC1
Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc. 855
PLCb is required for maximum amino acid-induced activity.
Increased intracellular calcium is necessary for amino acid-
induced mTOR activation in HeLa cells (Gulati et al., 2008). We
found that T1R1/T1R3 was necessary for amino acid-induced
calcium influx and that this calcium influx was necessary for
optimal mTORC1 activation (Figure 5F). Consistent with a role
for calcium, blockade of L-type calcium channels interfered
with amino acid stimulation of mTORC1 in b cells (Figure 5G).
Our findings suggest that amino acids bind T1R1/T1R3 leading
to activation of PLCb, calcium entry, and ERK1/2, thus stimu-
lating mTORC1 (Figure 7J).
TSC2 and REDD1 are among negative regulators of mTORC1
activity (Brugarolas et al., 2004). We tested the idea that knock-
down of T1R3 might decrease mTORC1 activation by increasing
the expression of these negative regulators. In HeLa cells
we found instead that the opposite occurred: silencing T1R3
Figure 4. mTORC1 Activity Is Lower in Tissues from T1R3–/–Mice
(A–D) C57BL/6J wild-type (WT) mice or T1R3 knockout (KO) mice were fasted 12–15 hr before sacrifice. (A) Immunoblots to detect pmTOR or pS6 from the
indicated organs are shown. Refer to the S6K immunoblot in Figure 7I for protein normalization for the corresponding tissues. (B and C) Means of arbitrary units
(AU) of either pmTOR/S6K or pS6/S6K ± SEM from six wild-type and six T1R3 knockout mice. (D) aa concentrations were determined in serum isolated from
mouse blood collected by cardiac puncture. Data are mean ± SEM from three wild-type mice and three T1R3 knockout mice.
(E–G) C57BL/6J mice were either fed or fasted for 6 or 12 hr before sacrifice. Protein and RNA were isolated from leg muscles for immunoblotting (E and F) and
qPCR (G). Data are normalized to S6 protein (E and F) or 18S RNA (G), and mean ± SEM of a total of six fed and six fasted mice for 12 hr from three independent
experiments. *p < 0.05, two-tailed Student’s t test. See also Figure S4 and Tables S1 and S2.
T1R1/T1R3 Regulates mTORC1
856 Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc.
resulted in a decrease in TSC2 and REDD1 (Figures 6A and 6B).
These findings suggest that silencing T1R3 causes changes that
would be expected if cells were engaging mechanisms to
compensate for loss of mTORC1 activity. Because loss of
TSC2 increases mTORC1 activity, mTORC1 is less readily in-
However, knockdown of TSC2 in HeLa cells does not rescue
amino acid activation of mTORC1 if T1R3 is also knocked
down (Figures S5D and S5E). The ability of constitutively active
Rag GTPases to keep mTORC1 active in the absence of amino
acids was also tested. However, weobserved a similar decrease
in mTORC1 activity upon amino acid deprivation in both the
control cells and cellstransfected with activeRagB (Figure S5G).
On the other hand, mTORC1 was less inhibited by amino acid
removal in HeLa cells highly overexpressing active RasV12
than control cells (Figure S5F).
Figure 5. T1R1/T1R3-Induced Signaling to
Following the indicated treatments, cell lysates
were immunoblotted with designated antibodies.
(A) MIN6 cells in KRBH were stimulated with the
indicated concentrations of aa (0.253–13) for
2 min (ERK1/2) or 30 min (S6). (B) MIN6 cells in
KRBH were pretreated with 10mMU0126 and then
stimulated with 1X aa for the indicated times. (C)
H9C2 cells pretreated as in (B) were stimulated
with aa for 15 min. (D) H9C2 cells transiently ex-
pressing control siRNA1 or T1R1 siRNA1 were
incubated in KRBH prior to stimulation with 25 mM
glucose and/or aa, or with 20% FBS for 30 min. (E)
The PLC inhibitor U73122 (10 mM), the inactive
analog U73343 (10 mM), or vehicle was added
to MIN6 cells in KRBH 15 min prior to treatment
with aa for the indicated times. (F) MIN6 cells
stably expressing either control shRNA2 or T1R3
shRNA1 were loaded with fura-2 in KRBH before
aa stimulation for 30 min. Data are mean ± SEM of
the 340/380 values of three independent experi-
ments. (G) MIN6 cells in KRBH were pretreated
with vehicle, or 10 mM nifedipine (Nif) for 15 min
prior to addition of vehicle or aa for the indicated
times. Data from all panels are representative of
three independent experiments, except (D), one of
two. See also Figure S5.
Reduced T1R1/T1R3 Expression
Does Not Deplete Intracellular
To determine if impairing T1R3 affected
intracellular amino acid concentrations,
we compared amino acid concentrations
in lysates from control and T1R3 knock-
down cells (Figure 6D). The amounts of
most amino acids including glutamine
and the nonessential amino acids, in
particular b-branched amino acids, were
similar in control and knockdown cells.
The relative amounts of amino acids
were also comparable to those reported
in muscle and other tissues (Kalhan
et al., 2011). To determine if a defect could be observed as
a result of amino acid depletion, we also examined amino acids
in cells that had been deprived of and then refed with amino
acids (Figure 6C). The amounts of most amino acids were again
similar in knockdown and control cells. Among differences,
concentrations of aspartic acid, serine, glycine, lysine, and
proline after refeeding were significantly higher in the control
cells; however, there were no differences in the concentrations
of branched chain amino acids between the refed control and
T1R3 knockdown cells.
Glutamine taken up by solute carrier family 1 member 5
(SLC1A5) is exchanged for leucine and other essential amino
acids through a SLC3A2/SLC7A5 heterodimer. This exchange
israte limitingforactivationofmTORC1byessentialamino acids
and growth factors (Nicklin et al., 2009). To determine if knock-
T1R1/T1R3 Regulates mTORC1
Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc. 857
Figure 6. Silencing T1R3 Induces Compensatory Changes
(A) HeLa cells transiently transfected with scrambled siRNA, or T1R3 siRNA4, were either left in complete growth medium or placed in EBSS for the indicated
times before harvest. The means of TSC2/GAPDH from control siRNA or T1R3 siRNA-transfected cells in DMEM, KRBH, or EBSS for various times from a total of
ten separate samples from two independent experiments are Control (11.0 ± 0.7) and T1R3 siRNA (5.6 ± 0.8). p < 0.01.
(B) HeLa cells with or without T1R3 knocked down as in (A), were in complete growth medium or KRBH for 1 hr and 45 min before harvest; one sample was
stimulated for 1 hr with aa as indicated. Proteins were detected by immunoblotting. Numbers under lanes are ratios of TSC2/GAPDH (A) or REDD1/GAPDH (B).
The means ± SEM of REDD1/GAPDH are Control (5.7 ± 0.39) and T1R3 siRNA (1.9 ± 0.22). p < 0.01.
(C) H9C2 cells with orwithout T1R3silenced asabove inKRBH werestimulated withaafor 30min. Intracellularaaconcentrationsweredetermined.Dataare area
under the curve and are the means of three replicates ±SEM. *, **, and ***p < 0.05, 0.01, and 0.001, respectively.
(D) Relative intracellular aa concentrations were determined from H9C2 cells transiently transfected with either control siRNA1 or T1R3 siRNA1 and grown in
completegrowthmedium.Dataaregraphed asaboveandarethemean±SEMofthreesamples.AsnandCyswerebelowdetectionlimits.His,Arg,and Lyswere
detected in only one of two experiments. Their concentrations in T1R3 knockdown cells did not change (His), increased 1.3-fold (Arg), or increased 2-fold (Lys)
when compared to cells transfected with control siRNA.
(E) RNA from H9C2 cells with T1R3 knocked down was analyzed by qPCR. Data are mean ± SEM of three independent experiments. *p < 0.05 control siRNA
versus the indicated transcripts. *p < 0.05, **p < 0.01, and ***p < 0.001, two-tailed Student’s t test. See also Tables S1 and S2.
T1R1/T1R3 Regulates mTORC1
858 Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc.
cells, cells were deprived of amino acids for 1 hr and then
pretreated with insulin and glutamine for an additional hour
prior to addition of leucine (Figure S5H). Under these condi-
tions, loss of T1R3 impaired activation of mTORC1 by leucine.
Based on these analyses, reduced mTORC1 activity is unlikely
to be due to insufficient intracellular glutamine available for
In a microarray experiment (data not shown), we noted that
SLC1A5, the glutamine transporter, appeared to be upregulated
severalfold by loss of T1R3, as did SLC3A2, a subunit of the
transporter that exchanges glutamine for essential amino acids.
Quantitative PCR (qPCR) analysis confirmed that mRNAs en-
coding both transporters were increased by T1R3 knockdown
in H9C2 cells (Figure 6E), further suggesting that a decrease in
neither the expression nor functionality of these transporters
contributed to reduced amino acid activation of mTORC1 in
the knockdown cells. Other transporters were also upregulated
including SLC7A11, a glutamate-cystine antiporter, which is
induced by amino acid starvation (Sato et al., 2004), and
SLC16A1, a lactate/pyruvate monocarboxylic acid transporter.
SLC7A3, a cationic amino acid transporter, was upregulated
15-fold in the knockdown cells.
Reduced T1R3 Activates Autophagy
Amino acid deprivation increases the rate of autophagy (Yang
and Klionsky, 2010). Conversion of the ubiquitin-like protein
LC3-1 to its lipidated form (LC3-II) and accumulation of LC3 in
autophagosomes are measures of autophagy that are visibly in-
creased byamino acid deprivation (Figures 7A and 7B). Because
mTORC1 suppresses autophagy, we probed the impact of
receptor loss on this process. Knockdown of T1R3 in H9C2 or
HeLa cells cultured in either complete growth medium or in the
siRNA Control T1R3
Heart Sk Muscle Liver
WT KO WT KO WT KO
siRNA Control T1R3
Relative LC3-II (AU)
Relative Protein (AU)
Figure 7. T1R3 Depletion Induces Auto-
(A) H9C2 cells in complete growth medium or in
KRBH were permeabilized, and LC3 was detected
(B) LC3 punctae in z stack images from (A) were
quantitated as described in Supplemental Exper-
imental Procedures and are the mean ± SEM of
(C) H9C2 cells transfected with either control
nontarget siRNA1 or T1R3 siRNA1 were incubated
in complete growth medium, and LC3 was de-
tected as above.
(D) LC3 punctae in z stack images from (C) were
quantitated as described in Supplemental Exper-
imental Procedures and are the mean ± SEM of
(E) Lysates of H9C2 cells that had been trans-
fected with the indicated siRNA were incubated in
KRBH for 2 hr before lysis. Lysates were im-
munoblotted to detect the indicated proteins.
(F) Quantitation of immunoblots is graphed as
ratios of the mean ± SEM of LC3-II/GAPDH of
a total of four samples from three independent
(G) H9C2 cells were transfected with the indicated
siRNA and cultured in complete growth medium
before harvest, and immunoblotting was per-
(H) Immunoblots from (G) were quantitated and
graphed as the ratio ± SEM of the amounts of
indicated proteins divided by the amount of S6.
(I) C57BL/6 wild-type or T1R3 knockout mice were
fasted for 12 hr before sacrifice. The indicated
proteins were immunoblotted.
(J) Model. aa are sensed directly via T1R1/T1R3.
PLCb is then activated, and calcium influx
increases, activating ERK1/2 and RSK. These
kinases aid in activation of mTORC1 by phos-
phorylating Raptor and TSC2. Growth factors feed
into this pathway at multiple levels. T1R1/T1R3
may also positively regulate mTORC1 by affecting
the localization of Rag proteins. **p < 0.01 and
***p < 0.001, two-tailed Student’s t test. See also
T1R1/T1R3 Regulates mTORC1
Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc. 859
absence of amino acids induced the accumulation of LC3-II and
the translocation ofLC3 toautophagosomes (Figures 7C–7F and
S6A). mTORC1 negatively regulates autophagy by phosphory-
lating ULK1 S757 (Kim et al., 2011). We observed that ULK1
S757 phosphorylation was significantly reduced in T1R3 knock-
down cells in nutrient-replete conditions. AMP-activated protein
kinase (AMPK) positively regulates autophagy by phosphory-
lating ULK1 S555 (Egan et al., 2011). We found a decrease in
AMPK activity in T1R3 knockdown cells as measured by
decreased phosphorylation of AMPK substrates ULK1 S555
and acetyl-CoA carboxylase (ACC) (Figures 7G and 7H). Thus,
autophagy induced by T1R3 knockdown is apparently not due
to increased AMPK activity but rather decreased mTORC1
Autophagy is induced in mouse after 12–24 hr of fasting (Ezaki
et al., 2011). To determine if T1R3 loss exacerbates autophagy
caused by caloric restriction, we measured p62 levels in tissues
from wild-type or T1R3?/?mice after a 12–15 hr fast. Decreased
p62, which is sequestered on autophagosomes and degraded
in lysosomes, is an indicator of increased autophagic flux. We
found decreased p62 in tissues of T1R3?/?mice compared to
wild-type (Figures 7I and S6B), suggesting that loss of T1R3
increases autophagic flux.
We provide evidence that the GPCR T1R1/T1R3 is a para-
mount mammalian amino acid detector that provides the
earliest notice of amino acid availability from the extracellu-
lar milieu to essential intracellular energy-sensing mechanisms.
Although not previously linked to mTORC1, amino acid-sensi-
tive GPCRs are well known in mammals; yet, the majority
of evidence has implicated amino acid uptake, metabolism,
and other unspecified intracellular events in activation of
mTORC1. GPCRs participate in sensing nutrients including
glucose and amino acids in fungi (Xue et al., 2008). In yeast,
amino acid detection by the TOR system involves nutrient
transport proteins, most of which have little or no ability to
transport amino acids to the cell interior and, instead, function
as membrane receptors (Forsberg and Ljungdahl, 2001; Hundal
and Taylor, 2009). Our conclusion that T1R1/T1R3 detects
extracellular amino acids prior to their uptake and transmits
that information to mTORC1 suggests that sensing amino acids
at the plasma membrane by TOR is evolutionarily conserved in
Several observations suggest that cells attempt to com-
pensate for the perceived deficiency in amino acids resulting
from taste receptor knockdown. Receptor knockdown, even
under nutrient-replete conditions, increased autophagy, consis-
tent with perceived starvation. Although intracellular concentra-
tions of amino acids in knockdown and control cells were
comparable, cells also upregulated mRNAs encoding several
transporters following receptor knockdown, to increase amino
acid and monocarboxylic acid uptake and flux, as if these
were impaired. Upregulated mRNAs included those encoding
the transporters, SLC1A5 and SLC3A2, shown to be rate limiting
for mTORC1 activation, and SLC7A11 and SLC16A1, both of
which are induced by starvation (Nicklin et al., 2009; Sato
et al., 2004; Ko ¨nig et al., 2008). Upregulation of these trans-
porters upon depletion of T1R3 further supports the idea that
this GPCR is a key amino acid sensor. In HeLa cells knock-
down of this GPCR resulted in downregulation of two negative
regulators of mTORC1, consistent with efforts by the cell to
relieve repression and reactivate mTORC1. In b cells with stable
T1R3 knockdown, insulin content was the lowest immediately
following antibiotic selection but increased over the subsequent
months. These findings suggest that mechanisms instigated to
offset the translational blockade due to mTORC1 inhibition facil-
itate increased protein synthesis and processing. How cells
overcome the loss of this receptor is not yet defined. Compen-
sation could be due to upregulation of related class C GPCRs
that may be able to transmit amino acid signals to mTORC1,
suppression of autophagy, even though it remains elevated
over the basal rate in T1R3 knockdown cells, or through intracel-
lular actions of leucine, arginine, or b-branched amino acids, for
Amino acids induce association of mTORC1 with intracellular
membrane compartments. With signaling from T1R1/T1R3 inter-
rupted, mTORC1 displays neither the characteristic lysosomal-
immunostaining pattern nor strong localization with lysosomal
markers. Thus, this receptor is required to direct the amino
acid-dependent intracellular trafficking of mTORC1. Activation
of mTORC1 by high concentrations of serum or strong overex-
pression of V12 Ras was detected in receptor knockdown cells,
suggesting that mTORC1 activity can be stimulated. Whether
these effects actually reflect mechanistic bypass of actions of
this receptor or are due largely to the pathophysiological stimuli
remains a question. On the other hand, mTORC1 activity was
poorly stimulated by EGF in receptor knockdown cells, consis-
tent with the requirement for amino acids to support growth
factor signaling to mTORC1. It remains possible that T1R1/
T1R3 communicates asignal to mTORC1 thatmay beredundant
with certain serum factors but that is not provided well by indi-
vidual growth factors. These results conform to the expectation
that maximal mTORC1 activation requires a convergence of
pressure from multiple extracellular molecules.
Fasted T1R3 knockout mice have significantly less mTORC1
activity in both heart and skeletal muscle than wild-type mice.
We were unable to consistently observe a significant decrease
in mTORC1 activity between the livers of fasted T1R3 knockout
mice and wild-type mice. Liver mTORC1 activity may be more
resistant to fasting than in other tissues due to increased
intraorgan nutrient availability caused by higher amounts of liver
autophagy. In fact mTORC1 activity was higher in livers from
rats fasted for 48 hr than in fed controls (Anand and Gruppuso,
Our findings reveal that T1R1/T1R3 conveys information on
amino acid availability to mTORC1. The fact that other GPCRs
such as GPR40 mediate some actions of free fatty acids has
demonstrated that nutrients have the potential to regulate cell
signaling and metabolism at extracellular as well as intracellular
levels in mammals (Itoh et al., 2003). Because membrane recep-
tors are readily accessible to drugs, T1R1/T1R3 may be a
valuable therapeutic target not only for cancer but also for
protein-wasting and degenerative diseases including diabetes,
cachexia, and Alzheimer’s disease.
T1R1/T1R3 Regulates mTORC1
860 Molecular Cell 47, 851–862, September 28, 2012 ª2012 Elsevier Inc.
These procedures are described in Supplemental Experimental Procedures.
Sources of materials and antibodies are in Supplemental Experimental
Transfection of shRNA and siRNA
MIN6 cells were electroporated with shRNA constructs (Supplemental Exper-
imental Procedures) using the AMAXA electroporation system with solution V
and program G-016. Stable cell lines were created by incubating cells in
2.5 mg/ml puromycin for ?1 month and were subsequently maintained in
puromycin. H9C2 rat cardiac myocytes were transiently transfected with
either control nontarget siRNA (Sigma-Aldrich; # SIC001) or MISSION siRNA
directed against T1R1 (#1), T1R3 (#1 or #2) designed and made by Sigma-
Aldrich. HeLa cells were transfected with either siGENOME Non-Targeting
siRNA pool #1 (Thermo Scientific) or T1R3 (#3) MISSION siRNA. The siRNA
sequences are in Supplemental Experimental Procedures. H9C2 cells were
transfected with siRNA using 5–20 nM siRNA with Lipofectamine RNAiMAX
(Invitrogen) following the manufacturer’s protocol and used 48–72 hr later.
Cell Culture, Immunofluorescence and Image Analysis, Cap Binding
Assays, Immunoblotting, and Quantitation
These procedures are described in Supplemental Experimental Procedures.
Tissue Harvest, RNA Isolation, and cDNA Synthesis
The experimental animal protocols used in this study were approved by the
Institutional Animal Care and Use Committee and are listed in Supplemental
Amino Acid Analysis
The 70%–90% confluent H9C2 cells in 150 mm plates were washed two to
three times with 10 ml PBS. Cells lysed in 50% methanol were snap frozen
in N2(liquid) and thawed at 37?C. Freeze/thaw was repeated four times. Insol-
uble material was sedimented at 16,000 3 g for 10 min at 4?C. Amino acids
were measured in the supernatants. Amino acid amounts were normalized
to protein concentration, determined in pellets by the BCA method (Pierce),
to compare samples. Aliquots (50 ml) of 50% methanol cell extracts were
deproteinized with 50 ml 3% (w/v) sulfosalicylic acid (Sigma-Aldrich) in
0.02 N HCl. Supernatants were analyzed on an L-8900 Amino Acid Analyzer
(Hitachi, Tokyo) equipped with a 6.0 mm ID 3 40.0 mm PF-High Speed-
packed ion-exchange column, fronted by a packed ion-exchange guard
column, and using manufacturer reagents. Detection was by ninhydrin post-
Supplemental Information includes six figures, two tables, and Supplemental
Experimental Procedures and can be found with this article online at http://
We thank David Mangelsdorf, Steven Kliewer, Joseph Albanesi, Elliott Ross,
Gray Pearson (Department of Pharmacology), Michael White (Department of
Cell Biology), Ondine Cleaver (Department of Molecular Biology), James Bru-
garolas (Department of Internal Medicine), and Sylvia Vega-Rubin-de-Celis
from the Brugarolas Lab, Natalie Ahn (University of Colorado, Boulder, CO),
and members of the M.H.C. lab for comments and suggestions, Nizar Ghneim
crine pancreatic tissue were provided by the Islet Resource Facility supported
by the University of Alabama, Birmingham, Comprehensive Diabetes Center.
This work was supported by grants from the National Institutes of Health
(R01 DK55310 and R37 DK34128 to M.H.C. and R01 CA157996 to R.J.D.)
and the Robert A. Welch Foundation (I1243 to M.H.C.). During the majority
of this work, E.M.W. was supported by a mentor-based postdoctoral fellow-
ship from the American Diabetes Association, A-Y.L. was supported by
a training grant from the Cancer Prevention and Research Institute of Texas,
Received: August 22, 2011
Revised: June 14, 2012
Accepted: August 2, 2012
Published online: September 6, 2012
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