(Received for publication, December 6, 1995)
From the
In pancreatic cells, cyclic AMP-dependent protein kinase
regulates many cellular processes including the potentiation of insulin
secretion. The substrates for this kinase, however, have not been
biochemically characterized. Here we demonstrate that the glucose
transporter GLUT2 is rapidly phosphorylated by protein kinase A
following activation of adenylyl cyclase by forskolin or the incretin
hormone glucagon-like peptide-1. We show that serines 489 and 501/503
and threonine 510 in the carboxyl-terminal tail of the transporter are
the in vitro and in vivo sites of phosphorylation.
Stimulation of GLUT2 phosphorylation in
cells reduces the initial
rate of 3-O-methyl glucose uptake by
48% but does not
change the Michaelis constant. Similar differences in transport
kinetics are observed when comparing the transport activity of GLUT2
mutants stably expressed in insulinoma cell lines and containing
glutamates or alanines at the phosphorylation sites. These data
indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies
the rate of transport. This lends further support for an important role
of the transporter cytoplasmic tail in the modulation of catalytic
activity. Finally, because activation of protein kinase A stimulates
glucose-induced insulin secretion, we discuss the possible involvement
of GLUT2 phosphorylation in the amplification of the glucose signaling
process.
Basal intracellular cAMP levels are critically required to
maintain the glucose-dependent secretory activity of pancreatic
cells. This is evidenced by the poor secretory response of cell sorter
purified
cells, which have a low intracellular cAMP content.
Activation of adenylyl cyclase, however, restores a normal
responsiveness to elevations in extracellular glucose (Pipeleers et
al., 1985; Schuit and Pipeleers, 1985; Yada et al., 1993;
Wang et al., 1993). Furthermore, acute increases in
intracellular cAMP above basal levels can further strongly potentiate
glucose-induced insulin secretion. This occurs for instance following
nutrient ingestion when carbohydrates and fat induce the secretion by
gut endocrine cells of two peptide hormones, GIP (glucose-dependent
insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1)
(Creutzfeld and Nauck, 1992; Dupre, 1991). These hormones bind to
specific
cell G protein-coupled receptors that activate the
adenylyl cyclase pathway and strongly potentiate glucose-induced
insulin secretion (Thorens, 1992a, 1994; Usdin et al., 1993;
Widmann et al., 1994; Gremlich et al., 1995). The
role of cAMP on the glucose competence state of
cells and its
acute stimulatory effects on insulin secretion indicate that multiple
protein kinase A-dependent phosphorylation events are involved in the
modulation of the insulin secretory activity. The substrates for PKA (
)in
cells, however, have not been biochemically
characterized.
The glucose transporter GLUT2 belongs to the family
of facilitated diffusion hexose transporters and is specifically
expressed in tissues that carry large glucose fluxes such as intestine,
liver, the proximal part of the kidney nephron, and pancreatic
cells (Thorens, 1992b). In
cells, GLUT2 catalyzes the uptake of
glucose, which is the first step in glucose-induced insulin secretion.
Glucose is then phosphorylated by glucokinase and following its
metabolism it induces insulin secretion by triggering plasma membrane
depolarization, influx of calcium, and fusion of secretory granules
with the plasma membrane. In the normal physiological state, the
rate-limiting step in this process is the phosphorylation of glucose by
glucokinase (Meglasson and Matschinsky, 1986).
Recent evidence has
suggested, however, that GLUT2 may participate in the glucose signaling
process by a mechanism distinct from its role as a sugar transporter.
For instance, Newgard and collaborators stably transfected insulinoma
cell lines that had lost GLUT2 expression and glucose-induced insulin
secretion with GLUT2 or GLUT1. Only the GLUT2-expressing cells and not
the GLUT1-expressing cells recovered a glucose-dependent secretory
response, although the rate of glucose metabolism was identical in both
transfectants. Furthermore, when the effect of forskolin was tested on
the secretory activity of the transfected cells, the increment in
insulin secretion was higher in the GLUT2-expressing cells (Hughes et al., 1992, 1993; Ferber et al., 1994). Also,
Valera et al.(1994) observed that a 70-80%
reduction in GLUT2 expression in
cells of transgenic mice
expressing GLUT2 antisense RNA leads to a decreased insulin secretory
response and to diabetes. This suggests that even in conditions in
which transport activity should not be limiting for glucose metabolism,
cell diabetic dysfunctions develop. This therefore indicates that
GLUT2 may serve a signaling role in
cells distinct from its
transport activity.
So far no report on GLUT2 phosphorylation has been published. In the present study we demonstrate that both forskolin and GLP-1 induce protein kinase A-dependent phosphorylation of GLUT2. Using fusion protein constructs and the expression of transporter mutants in intact cells, we determined that the PKA-dependent phosphorylation sites were specific serine and threonine residues of the carboxyl-terminal tail of the transporter. Phosphorylation led to a change in the intrinsic activity of the transporter further supporting previous data (Oka et al., 1990; Katagiri et al., 1992) showing that the carboxyl-terminal tail of GLUTs are important in the modulation of the transport activity. Finally, we discuss the possibility that GLUT2 phosphorylation may participate in the potentiation of insulin secretion induced by activators of the adenylyl cyclase pathway.
For Western blot analysis of GLUT2 and GLUT2 mutants, cell lysates in 80 mM Tris-HCl, pH 6.8, 5% SDS, 5 mM EDTA, 0.2 mMN-ethylmaleimide, and 1 mM phenylmethylsulfonyl fluoride were mixed with Laemmli sample buffer containing 5% SDS and separated by electrophoresis on SDS-containing 7.5% polyacrylamide gels. After transfer to nitrocellulose filters, detection of GLUT2 was performed as described previously using the ECL system from Amersham Corp. (Thorens et al., 1993).
The carboxyl-terminal cytoplasmic tail of GLUT2 contains several potential sites for phosphorylation by protein kinase A (Fig. 1)(Fukumoto et al., 1988; Thorens et al., 1988; Suzue et al., 1989). Except for threonine at position 510, which is unique to the rat GLUT2 sequence, the other PKA sites are also present in mouse and human GLUT2.
Figure 1: Sequence of the carboxyl-terminal cytoplasmic tail of GLUT2 from the end of the 12 transmembrane domain. The serine and threonine residues that were shown to be phosphorylated by protein kinase A are labeled by arrows and are at positions 489, 501/503, and 510. The mutations introduced in GLUT2 and in fusion proteins (GST) are described below the wild-type sequence.
To determine
whether GLUT2 present in cells was phosphorylated in response to
elevations in intracellular cAMP, we labeled pancreatic islets with
radioactive phosphate and exposed them to CPT-cAMP and IBMX for
different periods of time. GLUT2 was then immunoprecipitated from cell
lysates and analyzed by gel electrophoresis (Fig. 2). A basal
level of GLUT2 phosphorylation was observed in the absence of stimuli
but was not increased by the drug treatment for periods from 1 to 10
min. Similar observations were made when forskolin or dibutyryl cAMP
were used instead of CPT-cAMP (not shown). The inability to increase
the phosphorylation of GLUT2 in intact islets may be due to the
presence of glucagon secreting cells. Indeed,
cells have
receptors for glucagon that are coupled to activation of adenylyl
cyclase (Van Schravendick et al., 1985; Jelinek et
al., 1993; Abrahamsen and Nishimura, 1995) and that may therefore
maintain a high intracellular cAMP level (Schuit and Pipeleers, 1985),
thus preventing further stimulation of PKA-dependent phosphorylation.
If this were the case, phosphorylation experiments performed on cell
sorter purified
cells should allow us to detect the stimulatory
effect of increases in cAMP on GLUT2 phosphorylation. Indeed, when
experiments were carried out with purified
cells, there was a low
basal level of GLUT2 phosphorylation that could however be stimulated
by the addition of forskolin or GLP-1 (Fig. 3A). The
quantification and summary of the data obtained in four different
experiments with purified
cells is presented in Table 1and
shows that GLP-1 was as potent as forskolin in inducing GLUT2
phosphorylation and that maximal phosphorylation was already obtained 5
min after the addition of the stimuli.
Figure 2: GLUT2 phosphorylation in intact pancreatic islets. Pancreatic islets were labeled with radioactive phosphate for 2.5 h, and CPT-cAMP (1 mM) and IBMX (1 mM) were added for the indicated periods of time (minutes). After cell lysis, GLUT2 was immunoprecipitated and separated by gel electrophoresis on 7.5% SDS-polyacrylamide gels. In the absence of stimulation (time = 0) a basal level of GLUT2 phosphorylation was detected that was, however, not increased by the addition of CPT-cAMP and IBMX.
Figure 3:
A,
GLUT2 phosphorylation in cell sorter purified cells.
cells
(5
10
) were labeled with radioactive phosphate for
2.5 h, and forskolin (FSK) at 100 µM or GLP-1 (10
nM) was added for the indicated periods of time in the
presence of IBMX. After cell lysis, GLUT2 was immunoprecipitated and
analyzed as in Fig. 2. A 5-10-fold stimulation of
phosphorylation was detectable (see Table 1). B, GLUT2
phosphorylation induced by GLP-1 in cell sorter purified
cells is
prevented by the protein kinase A inhibitor H-89.
cells (5
10
) were labeled with radioactive phosphate for 2.5
h, and the inhibitor H-89 at 10 µM was added to the cells
for the last hour of the radioactive labeling. Cells were then
stimulated or not by GLP-1 in the presence of IBMX for 5 min. GLUT2 was
immunoprecipitated and analyzed as described in the legend to Fig. 2. C, analysis of the phosphoamino acids from
GLUT2 immunoprecipitated from the
cell lysates. Phosphoserines
and phosphothreonines are detected.
To determine whether PKA was responsible for the observed phosphorylation, an experiment was carried out in the presence or the absence of the PKA inhibitor H-89 (Chijiwa et al., 1990). As shown in Fig. 3B, the presence of the inhibitor completely prevented the stimulation of GLUT2 phosphorylation. The phosphorylated amino acids present in GLUT2 were then determined by phosphoamino acid analysis. As shown in Fig. 3C, both phosphoserines and phosphothreonines were detected.
As a first approach to identify the phosphorylation sites, we prepared fusion proteins consisting of the glutathione S-transferase (GST) and either the middle intracellular loop (amino acids 237-301, GST-ML), which does not contain putative PKA sites, or the carboxyl-terminal cytoplasmic tail (amino acids 481-522, GST-CT) of GLUT2. These proteins were phosphorylated in an in vitro reaction in the presence of the catalytic subunit of protein kinase A and radioactive ATP. In Fig. 4A, the left panel shows the Coomassie Blue staining of the fusion proteins from the in vitro reaction. On the right panel, the autoradiogram of the same gel demonstrates that only the fusion protein containing the carboxyl-terminal region (GST-CT) was phosphorylated by PKA, consistent with the presence of putative PKA sites in the transporter carboxyl-terminal tail. By phosphoamino acid analysis, both phosphoserines and phosphothreonines were detected in the fusion protein (Fig. 4C).
Figure 4: In vitro phosphorylation of fusion proteins by the catalytic subunit of protein kinase A. Fusion proteins consisting of the GST protein and different segments of wild-type or mutated GLUT2 were incubated in the presence of PKA and radioactive ATP. Incorporation of radioactivity was determined after separation of the reaction mixture by gel electrophoresis and autoradiography. A, on the left is the Coomassie Blue staining of the different proteins: GST, GST-CT (CT indicates the cytoplasmic tail of GLUT2), and GST-ML (ML indicates the middle loop of GLUT2). The lower bands in the last two lanes probably represent prematurely stopped translation products. On the right is the autoradiogram of the same gel. Only the band corresponding to the fusion protein containing the cytoplasmic tail of GLUT2 was labeled. B, in vitro phosphorylation of GST fusion proteins containing mutated forms of the cytoplasmic tail of GLUT2 (see the legend to Fig. 1for a description). The upper panel is the Coomassie Blue staining of the gel and indicates that equal amounts of proteins were used in the phosphorylation reactions. Lower panels, short and long autoradiographic exposures of the Coomassie Blue-stained gel. C, analysis of the phosphoamino acids of the fusion proteins labeled in B.
To define the
phosphorylation sites, we generated four different variants of the
GST-CT fusion proteins with various combination of serine and threonine
mutations, as described in Fig. 1. Fig. 4B shows
in the upper panel the Coomassie Blue staining of the fusion
proteins and in the lower panel two different autoradiographic
exposures of the same gel. In Fig. 4C, the phosphoamino
acid maps of these fusion proteins are presented. From these
experiments one can deduce that Thr is the only threonine
phosphorylated because its mutation led to the disappearance of
phosphothreonine (Fig. 4C). With regards to serines,
mutation of Ser
led to a fusion protein that was
phosphorylated mostly on Thr but also, at a low level, on serines as
can be detected after longer exposure of the thin layer chromatogram.
Also, the GST-CT-SST-E fusion protein with mutation of Ser
and Thr
was still phosphorylated on serines (Fig. 4C). Additional mutations of Ser
(GST-CT-SSST-E) led to a protein that could no longer be
phosphorylated. Thus, Ser
, Ser
, and
Thr
are the in vitro sites of PKA
phosphorylation on the GLUT2 cytoplasmic tail.
To determine whether the phosphorylation sites identified with the fusion proteins were indeed those used in vivo, we transiently transfected Cos cells with wild-type GLUT2 or the SSST-E mutant. The cells were then labeled with radioactive orthophosphate and stimulated or not with forskolin in the presence of IBMX. As shown in Fig. 5, activation of adenylyl cyclase led to an increase in GLUT2 phosphorylation but not in the phosphorylation of the SSST-E mutant. These data thus indicate that the phosphorylation sites identified in vitro on fusion proteins are indeed used in intact cells by activated protein kinase A. Also, because a basal level of phosphorylation is observed in the absence of stimulation of adenylyl cyclase in both the wild-type and mutant forms, this indicates that there are one or more additional sites in GLUT2 that are phosphorylated by an as yet not identified kinase.
Figure 5:
Phosphorylation of GLUT2 and the SSST-E
mutant transiently transfected in Cos cells. Transfected Cos cells were
labeled with [P]orthophosphate and accumulation
of intracellular cAMP and activation of PKA were stimulated by the
addition of forskolin and IBMX for 15 min. The wild-type and mutated
transporters were then immunoprecipitated and separated on
polyacrylamide gels. Densitometry scanning analysis shows a 4.8-fold
stimulation of wild-type GLUT2 phosphorylation. This is a
representative experiment out of three
performed.
To determine whether
phosphorylation of GLUT2 in normal cells induced a change in the
kinetics of glucose uptake, cell sorter purified
cells were
stimulated or not with GLP-1 in the presence of IBMX in conditions that
give maximal phosphorylation of GLUT2. The cells were then used to
determine the initial uptake velocity in the presence of 20 mM 3-O-methyl glucose. As shown in Fig. 6,
pretreatment of the cells with GLP-1 led to a reduction in the initial
velocity by about 40%. Measurement of 3-O-methyl glucose
uptake over a range of concentrations (Fig. 6B) further
indicated that in the presence of GLP-1, the K
value for uptake (14.7 mM) was not significantly
different from that reported in preceding publications, (17-18
mM) (Johnson et al., 1990; Heimberg et al.,
1993, 1995). These data thus indicate a decrease in the catalytic
activity of the transporter upon phosphorylation.
Figure 6:
Initial rate of 3-O-methyl
glucose uptake by cell sorter purified cells. A,
cells were incubated for 20 min at 37 °C in the presence or the
absence of 10 nM GLP-1 and 1 mM IBMX, and the cells
were then used for uptake measurements performed at 15 °C in the
presence of 20 mM substrate. The pretreatment with GLP-1
decreased the initial velocity by about 48%. (5.8 ± 1.3 versus 3.0 ± 0.6 pmol/nl space/min, mean ± S.D.
of four experiments, each performed with quadruplicate determinations
of uptake; difference by paired two-tailed Student's t test, p = 0.0211). B, rate of
3-O-methyl glucose transport as a function of increasing
substrate concentrations by
cells pretreated with GLP-1. C, Eadie-Hofstee transformation of the data in B shows that a single transport component is detected with a V
of 7.8 pmol/nl/min and a K
of 14.7 mM. The data represent the mean of four
experiments. The regression coefficient of the Eadie-Hofstee plot was r = 0.9.
We next assessed
whether the presence of negative charges at the sites of
phosphorylation could result in a change of transport kinetics similar
to that observed with intact cells stimulated with GLP-1. We thus
constructed GLUT2 mutants in which the identified phosphorylation sites
were mutated either to alanine (GLUT2 SSST-A) or to glutamate (GLUT2
SSST-E). In these mutants, no PKA-dependent phosphorylation can take
place, and in the SSST-E mutant, the glutamate residues introduce
negative charges that may mimic the phosphorylated state of GLUT2, as
reported for the L-type calcium channel for instance (Li et al., 1993). Both transporter mutants were stably
transfected in RINm5F cells. Selected clones were then used in glucose
uptake experiments to determine K
and V
. Total expression of GLUT2 in these cell lines
was determined by Western blot analysis, and surface expression of the
transporter was verified by a trypsin assay performed at 4 °C
(Thorens et al., 1993). Fig. 7A shows by
quantitative Western blot analysis that both cell lines expressed
identical level of transporters and that the transporters were present
only at the cell surface. Fig. 7B shows the
concentration-dependent increase in initial uptake rates by the SSST-A-
and SSST-E-expressing insulinoma cells and the Eadie-Hofstee
transformation of the data. Because both cell lines expressed identical
amounts of transporters, the V
for both mutants
can be directly compared from the Eadie-Hofstee plots. Table 2presents a summary of the K
and V
values determined in four separate
experiments. Altogether the data demonstrate that the mutations of the
phosphorylation sites into glutamates induced a
40% decrease in
the V
for 3-O-methyl glucose uptake
compared with the alanine mutant. The difference in the K
values for both mutants, although significant
when compared by t test analysis, is rather low. These data
compare well with the uptake measurements made with cell sorter
purified
cells. Similar data have been obtained by comparing
another set of GLUT2 SSST-A and SSST-E mutants.
Figure 7:
3-O-Methyl-glucose uptake by
insulinoma cells expressing either the SSST-E or SSST-A GLUT2 mutants. A, quantitation and surface expression of both mutants in
transfected insulinomas. Left, total cellular expression of
SSST-E and SSST-A mutants in the cell lines used for uptake
measurements as determined by Western blot analysis on increasing
concentrations of cell lysates (in µg) as indicated. Duplicate
determinations are shown. Right, the surface expression of the
mutated transporters was confirmed by trypsin treatment of the cells at
4 °C, which converts GLUT2 into a faster migrating form. B, uptake was measured as described under ``Material and
Methods.'' In the left panel is the rate of uptake by
SSST-E (closed squares) and SSST-A (open circles) as
a function of increasing substrate concentrations. Each point is the
mean of three measurements. In the right panel is the
Eadie-Hofstee transformation of the uptake data. Calculated V and K
are 493
nmol/min/mg protein and 26 mM, respectively, for SSST-A and
305 nmol/min/mg protein and 22.5 mM, respectively, for SSST-E
(see also Table 2).
Phosphorylation of GLUT2 in cell sorter purified cells
was shown to be a rapid event initiated either by direct activation of
adenylyl cyclase with forskolin or by GLP-1 binding to its receptor.
That phosphorylation of the transporter was protein kinase A-dependent
was further demonstrated by the inhibition of GLP-1-induced
phosphorylation by the specific protein kinase A inhibitor H-89.
Several sites of phosphorylation were identified in the cytoplasmic
carboxyl-terminal tail of GLUT2 using fusion proteins phosphorylated in vitro by the catalytic subunit of protein kinase A. That
the same sites are also substrates for PKA in intact cells was further
demonstrated by expressing the wild-type GLUT2 and the mutant lacking
the identified phosphorylation sites in Cos cells. Only wild-type GLUT2
phosphorylation can be increased upon stimulation of adenylyl cyclase
with forskolin. In these experiments, however, the SSST-E mutant was
still phosphorylated to a low basal level, indicating that the
transporter is phosphorylated on an additional site by a kinase
different from protein kinase A that has not yet been identified.
3-O-Methyl glucose uptake experiments performed on cell
sorter purified cells in the presence or the absence of GLP-1
indicated that under conditions that induce maximal phosphorylation of
GLUT2, there was a
48% reduction in the initial uptake velocity
measured in the presence of 20 mM substrate. This
concentration of 3-O-methyl glucose is slightly above the K
of the transporter. These experiments are
difficult to perform at higher substrate concentrations because the
number of cells is a limiting factor in these experiments and with more
diluted radioactive tracers the accuracy of the data decreases.
Nevertheless, these data provide indications that a consequence of
GLUT2 phosphorylation is a reduction of its catalytic activity.
Furthermore, we know from previous experiments (Thorens et
al., 1993) that GLUT2 is permanently expressed at the cell surface
even in intact islets in which the basal level of phosphorylation is
elevated. Thus stimulation of GLUT2 phosphorylation is not accompanied
by internalization of the transporter, and decrease in initial uptake
rate cannot be accounted for by a decreased cell surface expression of
the transporter.
Uptake experiments conducted with mutated
transporters expressed in insulinoma cells not expressing the
endogenous GLUT2, indicated that the mutant with glutamate residues in
place of serines 489 and 501/503 and threonine 510 had a V reduced by
40% compared with the alanine
mutant. These data very closely correlate with the observed effect of
phosphorylation of GLUT2 in
cells and strongly suggest that
introduction of negative charges by substitution of serines and
threonines by the acidic amino acids glutamate mimics the
phosphorylated state of the transporter. Similar conclusions have also
been drawn, for instance, in the case of the L-type Ca
channel (Li et al., 1993).
When glucose uptake is
measured using dispersed islet cells, two transport components are
observed with values of 17 mM and 1-2 mM (Johnson et al., 1990; Heimberg et al., 1993).
In purified
cells, only the high K
component
is observed (Heimberg et al., 1995). Because in purified
cells GLUT2 is only very poorly phosphorylated, whereas in dispersed
islets cells that contain glucagon cells its level of phosphorylation
may be higher, we hypothesized that the phosphorylated form of GLUT2
may account for the presence of the low K
transport component. However, this is not supported by the
present experimental evidences. Indeed, the K
for
3-O-methyl glucose uptake measured upon stimulation of
cells with GLP-1 had a value not very different from values published
previously (14.8 mMversus 17-18 mM)
(Johnson et al., 1990; Heimberg et al., 1993, 1995).
Experiments performed with the glutamate mutant expressed in insulinoma
cells led to the same conclusion. Indeed, even if the decrease in K
was statistically significant, it was small and
could not account for the low K
transport
component observed in dispersed
cells.
How can phosphorylation
of the cytoplasmic tail affect transporter activity? Glucose
transporters facilitate the diffusion of glucose molecules across
biological membranes by a mechanism probably best described by the
alternating conformer model. In this model the transporter has two
mutually exclusive sugar binding sites, one present on the
extracellular and the other on the intracellular surface. Binding of
glucose to one site induces the transporter to switch to the opposite
conformation, a process that is accompanied by movement of the
substrate across the membrane (reviewed in Carruthers(1990)). The
molecular basis for this conformational change is not yet elucidated
(Gould and Holman, 1993). However, reports have suggested that the
carboxyl-terminal cytoplasmic tail may be important in determining the
glucose transport function. For instance, deletion of this region in
the structurally related GLUT1 transporter isoform generated a
transporter that was locked in an inward facing conformation and was
thus unable to transport glucose (Oka et al., 1990).
Furthermore, replacing the cytoplasmic tail of GLUT1, a low K (1-3 mM) high affinity
transporter, with that of GLUT2 generated a chimera with increased V
and K
(Katagiri et
al., 1992). In the GLUT4 transporter isoform phosphorylation in
the cytoplasmic tail at serine 488 has also been reported to affect the
intrinsic activity of the transporter (Reusch, 1994), although this
view has been challenged (Lawrence, 1994). The present identification
of PKA phosphorylation sites at serines 489 and 501/503 and threonine
510 and their role in decreasing the catalytic activity of the
transporter further support an important role for the cytoplasmic tail
in the control of transporter function. We further show that this
process can be acutely regulated by hormones.
What is the possible
role of GLUT2 phosphorylation on cell secretory activity? In the
normal physiological situation, the rate of glucose transport in
cells is 50-100-fold in excess over the rate of phosphorylation.
GLUT2 thus appears to play mainly a permissive role, allowing an
unrestricted access of glucose to glucokinase. A
40% decrease in V
should therefore not impair the functioning of
the glucose sensor. In addition, because GLP-1 stimulates insulin
secretion, it cannot be easily understood how a decreased intrinsic
activity could participate in the stimulatory effect. The possibility
that GLUT2 may have an effect on the stimulation of insulin secretion,
beside its transport function, has been proposed by Newgard and
collaborators (Hughes et al., 1992, 1993; Ferber et
al., 1994). When they transfected insulinomas with GLUT2 but not
GLUT1, the cells recovered a glucose-dependent secretory response,
although the rate of glucose metabolism was identical in both
transfectants. Furthermore, when the effect of forskolin was tested on
the secretory activity of the transfected cells, the increment in
insulin secretion was higher in the GLUT2-expressing cells. Also,
Valera et al.(1994) observed that a
70-80%
reduction in GLUT2 expression in
cells of transgenic mice
expressing GLUT2 antisense RNA leads to a decrease insulin secretory
response and to diabetes. This suggests that even in conditions in
which transport activity should not be limiting,
cell diabetic
dysfunctions develop. These data therefore suggest that GLUT2 may serve
a signaling role in
cells distinct from its transport activity.
What is this signaling activity? Although mostly speculative at the
moment, one can suggest that phosphorylation of GLUT2 may promote
interactions with other protein components involved in the glucose
signaling pathway. Phosphorylation of membrane receptors or associated
proteins has been shown to be essential for several hormone or growth
factor signaling pathways. The characterization of the phosphorylation
sites and the generation of fusion proteins containing the variously
mutated form the transporter cytoplasmic tail may help identify
proteins normally interacting with GLUT2. This could for instance be
performed by testing the specific absorption of biosynthetically
labeled islet proteins with the fusion proteins (Cosson and Letourneur,
1994) by screening of cDNA expression libraries with the fusion
proteins (Skolnis et al., 1991) or by the yeast double hybrid
system using the glutamate or alanine forms of the transporter
cytoplasmic tail (Fields and Song, 1995; Chien et al., 1991).