(Received for publication, June 23, 1995; and in revised form, February 6, 1996)
From the
Turnover numbers for 3-O-methylglucose transport by the homologous glucose transporters GLUT1 and GLUT4 were compared to those for truncated and chimeric transporters expressed in Xenopus oocytes to assess potential regulatory properties of the C-terminal domain. The ability of high intracellular sugar concentrations to increase the turnover number for sugar entry (``accelerated exchange'') by GLUT1 and not by GLUT4 was maintained in oocytes. Replacing the GLUT1 C terminus with that of GLUT4 stimulated turnover 1.6-fold, but abolished accelerated exchange. Thus, the GLUT1 C terminus permits accelerated exchange by GLUT1, but in doing so must interact with other GLUT1 specific sequences since the GLUT4ctrm1 chimera did not exhibit this kinetic property. Removal of 38 C-terminal amino acids from GLUT4 reduced its turnover number by 40%, whereas removing only 20 residues or replacing its C terminus with that of GLUT1 increased its turnover number 3.5-3.9 fold. Therefore, using mechanisms independent of those which alter transporter targeting to the plasma membrane, C-terminal mutations in either GLUT1 or GLUT4 can activate transport normally restricted by the native C-terminal domain. These results implicate the C termini as targets of physiological factors, which through covalent modification or direct binding might alter C-terminal interactions to regulate intrinsic GLUT1 and GLUT4 transporter activity.
GLUT1 and GLUT4 are two members of a larger family of facilitative glucose transporters. GLUT1 is expressed in most cell culture lines and in many tissues, whereas GLUT4 is restricted primarily to muscle and adipose tissue (for review, see (1) ). Two factors determine the relative effectiveness by which GLUT1 or GLUT4 catalyze sugar transport: (a) the extent to which each transporter is targeted to the cell surface, and (b) their kinetic properties once resident in the plasma membrane. The amino acid sequences of the two transporters share 66% identity and 13% similarity, while non-conservative differences are localized to five domains, which include the N terminus, the large extracellular loop, a portion of transmembrane domains II and III, the large cytosolic loop, and the C terminus (see Fig. 1). An understanding of which of these domains account for the unique targeting and kinetic properties that distinguish GLUT1 and GLUT4 behavior is evolving rapidly.
Figure 1:
A
comparison of the amino acid sequences of mouse GLUT1 and mouse GLUT4
facilitative glucose transporters. A, diagram of amino acids
1-509 of GLUT4 showing the 12 transmembrane domains, the
cytosolic C-terminal and N-terminal domains, and the large cytosolic
and extracellular loops that characterize the predicted topology of
GLUT1 and GLUT4 transporters. Identical, similar, or non-conservative
sequence differences between GLUT1 and GLUT4 are shown. B, a
comparison of amino acid residues 402-492 of GLUT1 and
418-509 of GLUT4, which comprise transmembrane domains XI and XII
and the C-terminal domain of GLUT1 and GLUT4 outlined(- - -) in panel A. The HphI site at which the C-terminal
domains were interchanged to form the GLUT1ctrm4 and GLUT4ctrm1
chimeras is marked, as are sites of stop codons introduced to form the
truncated transporters GLUT1M1, GLUT4M1, and GLUT4M2. Also marked are
the amino acid identities () and similarities (
) between
the GLUT1 and GLUT4 sequences. The shaded region designates a
C-terminal ``core'' of amino acids common to GLUT1 and GLUT4,
which are required for maximal rates of sugar
transport.
Cellular control of GLUT4 transporter targeting to internal or plasma membrane compartments is a major mechanism for regulating glucose transport in insulin responsive tissues. GLUT4, unlike GLUT1, contains both an N-terminal internalization sequence, which increases the efficiency of retrieval of GLUT4 relative to GLUT1 from the plasma membrane(2) , and a C-terminal sequence, which restricts GLUT4 to intracellular sites if insulin is absent(3, 4, 5, 6) . When insulin is present, a higher proportion of intracellular GLUT4 than GLUT1 is redistributed to the plasma membrane to facilitate glucose transport across the plasma membrane(7, 8, 9, 10, 11) .
The turnover numbers for sugar transport by GLUT1 and GLUT4 are
roughly comparable(12, 13) , while the K for GLUT1 is typically higher than that
of
GLUT4(12, 13, 14, 15, 16, 17) .
GLUT1 demonstrates a unique kinetic property of ``accelerated
exchange'' in which the V
for transport of
sugar into a cell or vesicle is much higher when measured under
equilibrium exchange than under ``zero-trans'' conditions
when little or no intracellular sugar is
present(15, 18, 19, 20, 21, 22) .
The stimulation of sugar influx by a high intracellular concentration
of glucose does not require translocation of additional transporters to
the plasma membrane. Rather, it occurs through an increase in the rate
constant for conversion of the transporter from an inward to outward
facing conformation when the inward facing binding site becomes
occupied by sugar. This property is not observed with
GLUT4(16, 17) .
Both transporters, once resident in the plasma membrane, can exhibit different levels of activity, which are regulated by translocation-independent mechanisms(23, 24, 25, 26, 27, 28, 29) . The structural features of GLUT1 and GLUT4 that are required for these forms of regulation are not known, although several reports emphasize the importance of the C-terminal domain(26, 29, 30, 31) . To characterize transport regulatory features that might reside within the C termini, we have compared, using Xenopus oocytes, the kinetic parameters for sugar transport by cell-surface native and mutated GLUT1 and GLUT4 transporters in which the C-terminal domains were interchanged or truncated.
To verify correct translation of the mutant constructs, GLUT1, GLUT4, GLUT1ctrm4, and GLUT4ctrm1 messages were translated in vitro and the transporter products analyzed by SDS-PAGE. As anticipated, the mobility of each of the smaller GLUT1 and GLUT1ctrm4 translation products was faster than that for GLUT4 or GLUT4ctrm1 (Fig. 2A). Antibody RE01 recognized the C terminus of GLUT4 and GLUT1ctrm4, but not GLUT1 or GLUT4ctrm1, while antibody RE09 recognized the large extracellular loop of GLUT4 and GLUT4ctrm1 but not GLUT1 or GLUT1ctrm4. Antibody RE11 recognized the C terminus of GLUT1 and GLUT4ctrm1, but not GLUT4 or GLUT1ctrm4. In addition antibody against the large cytosolic domain of GLUT1 (RE18) recognized GLUT1 and GLUT1ctrm4, while antibody against the N terminus of GLUT4 (RE15) recognized GLUT4 and GLUT4ctrm1 (Fig. 3A). These results indicate correct translation of the constructs for native and chimeric transporters.
Figure 2:
Characterization of the translation
products of native, chimeric, and truncated mouse transporters. A, SDS-PAGE of [S]methionine-labeled
GLUT1, GLUT4, GLUT1ctrm4, and GLUT4ctrm1 after immunoprecipitation with
domain-specific antibodies RE01, RE09, and RE11. B, SDS-PAGE
of [
S]methionine-labeled GLUT4, GLUT4M1, and
GLUT4M2 translation products after extraction from Xenopus oocytes and immunoprecipitation with antibody RE15 against the N
terminus or antibody RE01 against the C terminus of
GLUT4.
Figure 3:
Membrane distribution and relative
turnover numbers for 3-O-methylglucose transport by native
(GLUT1 and GLUT4), truncated (GLUT4M1 and GLUT4M2), and chimeric
(GLUT4ctrm1 and GLUT1ctrm4) transporters expressed in Xenopus oocytes. A, an autoradiograph of
[S]methionine-labeled transporter proteins
separated by SDS-PAGE after immunoprecipitation from total or isolated
plasma membrane extracts. B, percentage of total oocyte
transporter protein recovered in the plasma membrane fraction. C, concentration of each transporter protein in isolated
oocyte plasma membranes normalized to the concentration of GLUT4. D, relative turnover number for sugar transport by each native
and mutant mouse transporter normalized to GLUT4. The number of
determinations (n) and the mean value obtained are shown above
each bar. Error bars represent the standard error of the
mean.
Transport properties determined for the native and chimeric
transporters expressed in Xenopus oocytes are presented in Table 1. Under zero-trans conditions, the V for uptake of sugar by native GLUT1 was 7.6 times that of native
GLUT4 and 2-fold that of GLUT1ctrm4. The V
for GLUT4ctrm1 was 6.3 times that of native GLUT4. No statistical
difference (p > 0.05) was observed between the K
values for zero-trans uptake of
3-O-methylglucose, which averaged 9.3 mM for both the
native and chimeric transporters.
In erythrocytes the V for sugar transport by GLUT1 under
equilibrium exchange conditions is typically higher than the
corresponding V
for sugar influx measured
when the intracellular concentration of sugar is
low(20, 21, 22, 42) . This property
is qualitatively retained by the mouse GLUT1 transporter when expressed
in Xenopus oocytes (Table 1). At 22 °C the V
for GLUT1 was 2.5 times the V
(p < 0.016), while the K
was 4.1 times the K
(p < 0.001). In
contrast, the V
and V
(influx) for transport by GLUT4 expressed in Xenopus oocytes were not statistically different (p > 0.05).
The K
for transport by GLUT4,
however, was significantly higher than the K
(p < 0.020).
Although the chimeric transporters GLUT1ctrm4 and GLUT4ctrm1
retained transport activity, replacement of the C terminus of GLUT1
with that of GLUT4 abolished accelerated exchange since the V and V
for
GLUT1ctrm4 were indistinguishable (Table 1). Replacement of the C
terminus of GLUT4 with that of GLUT1, however, did not confer
accelerated exchange upon the GLUT4ctrm1 chimera, since its values of V
and V
for
transport (like those for GLUT4 and GLUT1ctrm4) were statistically
indistinguishable. While the K
for
GLUT1 was significantly higher (p < 0.003), no significant
differences (p > 0.05) between the K
's for GLUT1ctrm4,
GLUT4ctrm1, and GLUT4 were observed. The GLUT1ctrm4 and GLUT4ctrm1
chimeras generated by the interchange, therefore, retained high
transport activity but did not retain or gain, respectively,
GLUT1's ability to exhibit accelerated exchange.
At 0.5
mM 3-O-methylglucose, the zero-trans rates of sugar
uptake by either native GLUT4 or truncated GLUT4M1 expressed in oocytes
were indistinguishable (p > 0.05), but both were
significantly higher than that of water injected oocytes (Table 1). Interestingly, the rate of 3-O-methylglucose
uptake by oocytes expressing GLUT4M2 was 3.6 times that of oocytes
expressing native GLUT4 (p < 0.04). The V for GLUT4M1 was 30% lower than that of
GLUT4 (p < 0.038), while the V
for GLUT4M2 was 4.5 times that of native GLUT4 (p <
0.001). The values of K
obtained
for GLUT4, GLUT4M1, and GLUT4M2 ranged between 7.6 and 9.3 mM,
but were not statistically different (p > 0.05). Thus,
without altering K
, removal of 38
C-terminal amino acids of GLUT4 only minimally reduces transport
activity, while removal of 20 amino acids dramatically increases
transport rates.
The data in Fig. 3B do not provide an accurate comparison of the
relative plasma membrane concentrations for each transporter, since the
total amounts of each expressed transporter protein were comparable,
but not identical. Rather, the transporter concentrations in the plasma
membrane were calculated as described under ``Materials and
Methods'' using the integrated intensities of the transporter
bands from isolated plasma membranes (Fig. 3A, right panel) and the concentration expressed relative to GLUT4 (Fig. 3C). These data provide the second internal
control to validate proper quantitation of each surface transporter.
The surface ratio (GLUT1/GLUT4) was established by Nishimura and
colleagues (13) both by isolating plasma membrane/vitalline
membrane complexes from [S]methionine-labeled
oocytes (as in this study) and by labeling GLUT1 and GLUT4 on the
surface of oocyte plasma membranes with ATB-BMPA. The ratio GLUT1/GLUT4
in the isolated plasma membranes determined in this study (Fig. 3C) was 3.5 and agrees well with the value of 3.8
determined by Nishimura et al. The observed difference in
ratio can be accounted for by at most an 8% contamination of the GLUT4
in the plasma membrane by GLUT4 from internal membrane sources. Thus,
the selected techniques effectively separate the plasma membrane from
internal vesicles containing GLUT1 or GLUT4 and reveal that the
concentrations of GLUT4M1, GLUT4M2, and GLUT1ctrm4 transporters in the
plasma membrane of Xenopus oocytes were not statistically
different than that of GLUT4 (p > 0.05), while those of
GLUT1 (GLUT1/GLUT4 ratio = 3.5) (p < 0.001) and
GLUT4ctrm1 (GLUT4ctrm1/GLUT4 ratio = 1.8) (p <
0.019) were significantly higher (Fig. 3C).
Figure 4:
Transport of 3-O-methylglucose by
functional GLUT1M1. A, uptake of 0.5 mM 3-O-methylglucose by Xenopus oocytes injected
with either water or with message encoding GLUT1M1. The ratio (n = 5) of the GLUT1M1 V to the
GLUT1 V
is shown in the inset. B,
autoradiograph of [
S]methionine-labeled GLUT1
and GLUT1M1 transporter proteins separated by SDS-PAGE after
immunoprecipitation from oocyte plasma membrane extracts. The ratio (n = 3) of GLUT1M1 to GLUT1 protein in the plasma
membrane was 0.62 ± 0.02. Values are presented as the mean
± standard error.
The C-terminal domains of GLUT1 and GLUT4 indirectly regulate cellular sugar uptake because they serve as recognition sequences for the cellular machinery that controls sorting to intracellular or plasma membrane sites. This study was initiated to determine whether these domains, apart from their sorting roles, might also function to regulate transport activity (turnover number) of transporters resident in the plasma membrane. The first 11 amino acids adjacent to transmembrane domain XII in the two C termini contain 1 similar and 10 identical amino acid residues (Fig. 1). Of the remaining C-terminal amino acids, 50% of the differences represent non-conservative changes. Thus, the C-terminal domains of GLUT1 and GLUT4 contain both conserved and divergent regions, which could confer either shared or unique regulatory properties to each of the transporters.
To examine the properties of the divergent sequences, the C-terminal domains were interchanged to alter, in effect, all dissimilar amino acids distal to Phe-444 (GLUT1) and Phe-460 (GLUT4). The resultant chimeric transporters GLUT1ctrm4 and GLUT4ctrm1 exhibited turnover numbers higher than those of the respective native GLUT1 and GLUT4 (Fig. 3D). The 4-fold higher turnover number for the GLUT4ctrm1 chimera could have arisen from a transport activating property of the GLUT1 C terminus, since GLUT1 in our study is more active than GLUT4. However, since the turnover number for the truncated transporter GLUT4M2 was also nearly 4 times that of the native transporter (Fig. 3D), a more reasonable interpretation for the turnover numbers for both GLUT4ctrm1 and GLUT4M2 being higher than GLUT4 is that in both instances an inhibitory domain has been removed, which includes all or a subset of amino acid residues 490-509 of GLUT4. Likewise, the higher turnover number observed for GLUT1ctrm4 arises from the removal of the inhibitory region present in the GLUT1 C terminus and not through activation by the GLUT4 C terminus, since the latter was derived from the less active GLUT4 transporter.
Both GLUT1 and GLUT4 C-terminal domains, therefore, suppress native transport capacity and for GLUT4 this inhibitory region has been localized to the C-terminal 20 amino acids. The physiological role of the inhibitory domain in GLUT4 is unclear. The C-terminal 29 amino acid residues of rat GLUT4 must be present for analogs of cAMP and AMP to inhibit GLUT4 transport in CHO cells, and transfer of this domain to GLUT1 generated a GLUT1ctrm4 chimera, which acquired nucleotide analog-induced inhibition of sugar transport(29) . It appears unlikely that C-terminal modifications in our study activate GLUT4 by relieving endogenous nucleotide-mediated inhibition of GLUT4 in Xenopus oocytes, since the GLUT1ctrm4 chimera has a turnover number higher than GLUT1, and the 3.5-3.9-fold activation observed by disrupting this domain in GLUT4 is much larger than the 50% inhibition of GLUT4 transport induced by nucleotide analogs in CHO cells(29) . The C-terminal domain of GLUT4 may in fact serve a dual function to facilitate either transport activation by unknown regulatory factors or transport inhibition in response to rising levels of certain nucleotides. The specific amino acids responsible for the inhibitory activity of the C-terminal domain of GLUT1 have been more difficult to define. Neither removal of the C-terminal 12 amino acids of rabbit GLUT1 (45) nor loss of the C-terminal 22 amino acids of human GLUT1 (46) significantly affects transporter activity. Therefore, the increase in the turnover number we observe by replacing the GLUT1 C terminus with that of GLUT4 might arise from the resultant change in 1 or more of the 9 non-identical GLUT1 amino acid residues that lie between amino acids 444 and 471, rather than from an alteration of the C-terminal 22 amino acids of GLUT1.
We have demonstrated the respective ability and
inability of GLUT1 (15) and GLUT4 (16, 17) to
exhibit accelerated exchange in their native cell environments is
retained when the two transporters are expressed in Xenopus oocytes. Therefore, the turnover number for sugar entry catalyzed
by GLUT1 can be elevated 2.5-fold (V/V
=
2.5; Table 1) or higher (46) in the oocyte environment
simply by raising the intracellular sugar concentration. No previous
reports to our knowledge have identified regions of amino acid
diversity that account for such ``accelerated exchange''
kinetics exhibited by GLUT1 and not by GLUT4. This study demonstrates
that replacing the C-terminal domain of GLUT1 with that of GLUT4
abolishes accelerated exchange. More importantly, this kinetic property
is not transferred with the C-terminal domain of GLUT1 when it replaces
that of GLUT4. Apparently, a prerequisite for accelerated exchange
kinetics is the ability of unique sequences within the C-terminal
domain of GLUT1 to interact specifically with one or more unique
sequences in another domain of GLUT1. This might occur directly or in
combination with other regulatory factors to establish a low rate of
conversion of the transporter from the inward facing to the outward
facing conformation by forming an energy barrier to changes in
transporter conformation when the inward facing site of the transporter
is unoccupied by sugar. These restrictive interactions, which
presumably are disrupted to activate transport when internal sugar
concentrations are raised, might be the same interactions that are
destroyed when the GLUT1 C terminus is replaced by that of GLUT4 to
simultaneously activate transport and abolish accelerated exchange.
The divergent sequences of GLUT1 and GLUT4, therefore, express differences in the degree to which they suppress transport and their involvement in accelerated exchange. Do the regions of amino acid identity in the C termini impart a common trait to GLUT1 and GLUT4? The amino acids in a ``core'' region common to both GLUT1 and GLUT4 (Fig. 1B, shaded area) have been considered essential to GLUT1 function. While the loss of 12 or 22 C-terminal amino acids from GLUT1 appears inconsequential, removal of the entire C-terminal 42 amino acids destroys >95% of the activity(46) , and loss of the C-terminal 37 amino acids of rabbit GLUT1 totally inactivates the transporter by locking it into an inward facing conformation(30, 44) . Our observations indicate that one or more of the identical amino acids and not the dissimilar amino acids within residues 457-468 of GLUT1 or 473-484 of GLUT4 are critical for normal transporter function since interchange of the C termini of mouse GLUT1 and GLUT4 only enhances sugar turnover. Although the high amino acid identity within the ``core'' region would predict that truncation of GLUT4 at the same site as rabbit GLUT1 should totally inactivate mouse GLUT4, this was not observed. The turnover number for GLUT4M1 remained 60% that of native GLUT4. Since this result was unexpected, we reevaluated the effect of truncation of GLUT1 at Lys-456 on transport activity using the mutant mouse GLUT1M1 and determined the turnover number for mouse GLUT1M1 to be >20% that of native mouse GLUT1. It is unclear why such subtle species differences should render the truncated rabbit mutant totally inactive yet allow the truncated mouse mutant to retain significant activity. Perhaps one or more of the 17 amino acid residues dispersed throughout the rabbit transporter which differ from those of the mouse transporter render the rabbit isoform more sensitive to C-terminal truncation within this region. Is this ``core'' region of amino acid identity therefore less important to GLUT4 transporter function since GLUT4M1 retains 60% of the transport capacity of native GLUT4? Apparently it is not. A better appreciation of the common role of this region in GLUT1 and GLUT4 arises when comparing the turnover number of GLUT4M1 to that of GLUT4M2. When the activated mutant of GLUT4 (GLUT4M2) is truncated further to form GLUT4M1, 84% of GLUT4M2 turnover capacity is lost. This is comparable to the 77% loss of GLUT1 activity observed after truncation to form the mutant GLUT1M1. Thus the common amino acids distal to Lys-456 of GLUT1 and Arg-472 of GLUT4 are not absolutely essential for function in the respective transporters, but do permit each transporter to express a 4-5-fold higher turnover capacity. Truncated GLUT4M1 appears nearly as active as native GLUT4 only because of the presence in GLUT4 of the C-terminal inhibitory domain, which restricts the full transport potential of native GLUT4.
In summary, the C-terminal domains of mouse GLUT1 and especially mouse GLUT4 limit the maximal transport capacity of the respective transporters in which they reside. Stimulation of GLUT4 activity can occur either by removing 20 amino acids from the C terminus or by replacing the GLUT4 C terminus with that of GLUT1. Higher transport rates by GLUT1 can be induced simply by raising the internal sugar concentration or by replacing the C-terminal domain with that of GLUT4. Each of these methods of activation have relatively little physiological basis, but share the common property that they may serve to alter interactions of the native C-terminal domain with other domains of the transporter and/or with other cytosolic proteins or factors which serve to regulate transporter activity. This is especially relevant in view of the recent identification of proteins capable of binding directly to fusion proteins containing either GLUT1 or GLUT4 C termini(47) . Several physiologically relevant examples of transport regulation which occur in cells by mechanisms independent of GLUT1 or GLUT4 redistribution were cited in the introduction. Perhaps these forms of activation target and alter C-terminal domain interactions to shift the transporters from a less to more active state as were accomplished by the mutations. Cited studies and our results emphasize the importance of investigating further the potential role of the C-terminal domain as a target for physiological mechanisms that regulate sugar turnover through activation of the transporter resident in the plasma membrane, an important form of regulation separate from the well documented activation of cellular sugar uptake via transporter redistribution.