From the Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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To study the role of the GTPase dynamin in GLUT4
intracellular recycling, we have overexpressed dynamin1 wild type and
a GTPase-negative mutant (K44A) in primary rat adipose cells.
Transfection was accomplished by electroporation using an hemagglutinin
(HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4
alone, insulin results in an
7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of
HA-GLUT4-trafficking parallel those of the native GLUT4 and in
addition, that newly synthesized HA-GLUT4 goes to the plasma membrane
before being sorted into the insulin-responsive compartments. Short
term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the
amount of cell surface HA-GLUT4 in both the basal and
insulin-stimulated states. Under conditions of maximal expression of
dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears
to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose
transporters. In contrast, expression of dynamin wild type decreases
the amount of cell surface HA-GLUT4 in both the basal and
insulin-stimulated states. These data demonstrate that the endocytosis
of GLUT4 is largely mediated by processes which require
dynamin.
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INTRODUCTION |
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In adipose cells, GLUT4 glucose transporters are constantly recycling between an intracellular compartment and the plasma membrane (1-4). In the basal state, where the rate of exocytosis is relatively low, the vast majority of the GLUT4 glucose transporters reside in an as yet poorly characterized intracellular compartment (1, 3, 5). Stimulation of adipose cells with insulin leads to an increase in the rate of exocytosis of GLUT4-containing vesicles, resulting in a rapid shift in the steady state distribution of GLUT4 to the plasma membrane (1-3). After clearance of the hormone, the rate of GLUT4 exocytosis decreases and the steady state distribution shifts back to the intracellular compartment.
The primary focus of recent investigations has been the identification and characterization of signaling molecules (e.g. p85/p110 phosphatidylinositol 3-kinase) and other cellular components (e.g. soluble NSF attachment protein receptors (SNAREs)) possibly involved in the regulated exocytosis of GLUT4 (6-11). However, little is known about the mechanism of GLUT4 endocytosis. Previous reports provided indirect evidence that GLUT4 might be internalized by a mechanism involving clathrin-mediated endocytosis. Potassium depletion, known to disrupt formation of clathrin-coated vesicles (12), results in a decreased internalization of GLUT4 and mannose-6-phosphate receptors in rat adipose cells (13). In 3T3-L1 adipocytes, GLUT4 has been shown to co-purify with clathrin-coated vesicles derived from the plasma membrane after treatment of the cells with the fungal toxin brefeldin A (14). Previous morphological analysis showed association of GLUT4 with clathrin-coated pits (4), whereas little co-localization of GLUT4 with clathrin is observed in a recent study from our laboratory (5). Since no functional studies involving components of clathrin-mediated endocytosis in insulin target cells have been reported, the mechanism of GLUT4 internalization still remains unclear.
The dynamins belong to a family of 100-kDa GTPases that
mediate the initial stages of endocytosis (15-18). To date, three
mammalian dynamin genes (referred as dynamin1 to dynamin
3) have
been described (15, 19-21). Dynamin
1 is found in neurons, dynamin
2
is expressed ubiquitously, and dynamin
3 is enriched in testis (15,
19-21). Although the distinct functions of these different dynamin
proteins are not fully understood, considerable evidence now indicates that dynamin
1 participates in clathrin-mediated endocytosis. Dynamin
1 colocalizes with clathrin in intact cells on the light and
electron microscopy levels (22, 23), and binds
-adaptin, a component
of clathrin-coated pits, in vitro (24). Furthermore, transfection of cultured mammalian cells with dominant-negative dynamin
1 mutants results in the accumulation of clathrin-coated pits
at the plasma membrane (18, 22), whereas internalization of transferrin
receptors, epidermal growth factor receptors, and
2-adrenergic
receptors is inhibited (17, 18, 22, 25). Even though a role of
dynamin
2 in endocytosis remains unclear, a recent report suggests
that dynamin
2 might also be localized to coated pits on the plasma
membrane (26). Thus, considering the high degree of amino acid sequence
homology between dynamin
1 and dynamin
2 and the ability of
dynamin
1 mutants to inhibit receptor-mediated endocytosis even in
nonneuronal cells, both isoforms might act as functional homologues in
endocytosis in nonneuronal cells (17, 18, 22, 25). However, the finding that dynamin
2 localizes to vesicles in the Golgi complex (27-29) implies additional functions of this isoform as well. To characterize the mechanism of GLUT4 endocytosis, we have overexpressed a
dominant-negative mutant of dynamin
1 in isolated rat adipose cells.
The effects of dynamin
1 on GLUT4- trafficking in vivo were
monitored by utilizing a co-transfected recombinant GLUT4 containing an
HA1 epitope tag in the first
exofacial loop (30).
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructs--
All constructs were generated in the
pCIS2 mammalian expression vector (a generous gift from Dr. C. Gorman).
cDNAs for HA epitope-tagged dominant-negative K44A dynamin1 and
HA-tagged wild-type dynamin
1 (a generous gift from Drs. H. Damke and
S. L. Schmid) were subcloned into the expression vector.
Construction of the HA-tagged GLUT4 has been described previously (30).
For transfection experiments, the plasmids were purified in mg
quantities using a maxiprep kit (Qiagen).
Cell Culture and Transfection of Rat Adipose
Cells--
Preparation of isolated rat epididymal adipose cells from
male rats (CD strain, Charles River Breeding Laboratories, Inc.) was
performed as described previously (31). Isolated cells were washed
twice with Dulbecco's modified Eagle's medium containing 25 mM glucose, 25 mM HEPES, 4 mM
L-glutamine, 200 nM
()-N6-(2-phenylisopropyl)-adenosine,
and 75 µg/ml gentamycin, and resuspended to a cytocrit of 40%
(
5-6 × 106 cells/ml). 200 µl of the cell
suspension were added to 200 µl of Dulbecco's modified Eagle's
medium containing 100 µg of carrier DNA (sheared herring sperm DNA;
Boehringer Mannheim) and expression plasmids as indicated. The total
concentration of plasmid DNA in each cuvette was adjusted to 5 µg/cuvette with empty pCIS2. Electroporation was carried out in
0.4-cm gap-width cuvettes (Bio-Rad) using a T810 square wave pulse
generator (BTX). After applying three pulses (12 ms, 200 V), the cells
were washed once in Dulbecco's modified Eagle's medium, pooled in
groups of 4-10 cuvettes, and cultured at 37 °C, 5% CO2
in Dulbecco's modified Eagle's medium containing 3.5% bovine serum
albumin.
Cell Surface Antibody Binding Assay--
Rat adipose cells were
harvested 3.5 or 20-24 h post-transfection and washed in Krebs-Ringer
bicarbonate HEPES buffer, pH 7.4, 200 nM adenosine (KRBH
buffer) containing 5% bovine serum albumin. Samples corresponding to
the cells from one cuvette were distributed into 1.5-ml microcentrifuge
tubes. After stimulation with 67 nM (1 × 104 microunits/ml) insulin for 30 min at 37 °C,
subcellular trafficking of GLUT4 was stopped by the addition of 2 mM KCN (32). All of the following steps were performed at
room temperature. A monoclonal anti-HA antibody (HA.11, Berkeley
Antibody Co.) was added at a dilution of 1:1000, and the cells were
incubated for 1 h. Excess antibody was removed by washing the
cells three times with KRBH, 5% bovine serum albumin. Then 0.1 µCi
of 125I-sheep anti-mouse antibody (Amersham Pharmacia
Biotech) was added to each reaction, and the cells were incubated for
1 h. Finally, the cells were spun through dinonylphtalate oil to
remove the unbound antibody (31), and the cell surface-associated
radioactivity was counted in a -counter. The resulting counts were
normalized to the lipid weight of the samples (31). Unless stated
otherwise, the values obtained for pCIS-transfected cells were
subtracted from all other values to correct for nonspecific antibody
binding. Antibody binding assays were performed in duplicate or
quadruplicate.
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RESULTS |
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Expression of HA-GLUT4--
To increase the insulin response of
rat adipose cells transfected with epitope-tagged GLUT4 above that
observed with the original technique (30), we tested the experimental
procedure as follows. Rat adipose cells were transfected with various
amounts of HA-GLUT4 expression plasmid and analyzed at different time
points for basal and insulin-stimulated cell surface HA-GLUT4 using the
anti-HA antibody binding assay. The tagged glucose transporters become detectable at the cell surface in response to insulin as early as
2 h post-transfection. Synthesis of HA-GLUT4 continues until about
16 h post-transfection, at which time its level stays relatively constant and maximal to 24 h post-transfection (data not shown). As judged by immunohistochemistry using the same monoclonal anti-HA antibody and nonpermeabilized, insulin-stimulated cells, the
electroporation procedure yields 10% HA-positive cells (Ref. 33 and
data not shown).
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Coexpression of Dynamins-- To study the effects of dynamin overexpression on the subcellular distribution of epitope-tagged GLUT4, rat adipose cells were co-transfected with HA-GLUT4 and various concentrations of expression plasmids for wild-type and mutant dynamin. Fig. 2 illustrates the data when protein expression is carried out for 3.5 and 20 h. At 3.5 h post-transfection in dynamin-K44A-transfected cells, the basal cell surface level of HA-GLUT4 was increased as much as 2.6 ± 0.1-fold (mean ± S.D.) compared with cells transfected with HA-GLUT4 alone (Fig. 2A). Likewise, a concomitant increase in cell surface HA-GLUT4 in the insulin-stimulated state was observed. (Fig. 2A). In contrast, expression of wild-type dynamin decreased the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states to 51 ± 4 and 58 ± 13% that of the controls, respectively. After 20 h of expression of the dynamin mutant, the basal level of cell surface HA-GLUT4 equaled that for the insulin-stimulated state (Fig. 2B). In addition, the absolute amount of cell surface HA-GLUT4 was increased by about 30-40% (37 ± 1% and 32 ± 12% in the absence and presence of insulin, respectively) compared with the insulin-stimulated control. Prolonged overexpression (20 h) of the wild-type dynamin decreased the amount of cell surface HA-GLUT4 in the insulin-stimulated state compared with both the control cells and the cells transfected with mutant dynamin and decreased the basal level compared with cells transfected with mutant dynamin (Fig. 2B), as shown for 3.5 h of expression (Fig. 2A).
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Time Course of HA-GLUT4 Internalization-- To verify that expression of the dynamins affects the endocytosis of GLUT4 in rat adipose cells, we analyzed the redistribution of cell surface HA-GLUT4 in the presence of the fungal metabolite wortmannin (7, 34, 35). Acting as an inhibitor of the lipid kinase phosphatidylinositol 3-kinase (36), wortmannin blocked the insulin-stimulated translocation of GLUT4 from its basal compartment to the plasma membrane (7, 35, 37). When added simultaneously with insulin, wortmannin (100 nM) also blocked translocation of epitope-tagged HA-GLUT4 to the cell surface in transfected cells (data not shown). Previously, Holman and co-workers (38) and a study from our laboratory (39) showed that this inhibition of GLUT4 translocation does not affect the early steps of GLUT4 endocytosis. Thus, the addition of wortmannin to insulin-stimulated cells inhibited further translocation of GLUT4 from its intracellular compartment to the plasma membrane, thereby allowing measurements of the kinetics of GLUT4 endocytosis. Fig. 3 demonstrates the results of such wortmannin experiments.
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Targeting of Newly Synthesized HA-GLUT4-- Evidently the protein synthesis-associated increase in cell surface glucose transporters in rat adipose cells utilizes a wortmannin-insensitive trafficking pathway. As shown in Fig. 3C, newly synthesized HA-GLUT4 still appeared on the cell surface in the presence of mutant dynamin and wortmannin. Under these conditions, the endocytosis of GLUT4 was inhibited by the dynamin mutant, leading to an accumulation of glucose transporters in the plasma membrane (Fig. 3E). Likewise, the translocation of GLUT4 from the intracellular pool to the plasma membrane was inhibited by wortmannin. To further investigate the site at which the newly synthesized glucose transporters enter their recycling compartments, we studied the effects of wortmannin on the cell surface level of HA-GLUT4 under basal conditions. To increase the antibody binding signal, the incubation time with wortmannin was extended to 2 h. The results are illustrated in Fig. 5.
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DISCUSSION |
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To study the subcellular trafficking of GLUT4 in an insulin target cell, we have transfected rat adipose cells with a recombinant glucose transporter containing an HA epitope tag in the first exofacial loop (30). The HA-GLUT4 was detected on the cell surface of transfected cells by the binding of an antibody against the HA epitope. The observed insulin response of HA-GLUT4 translocation to the plasma membrane is markedly reduced when the amount of expression plasmid is increased and/or the time period of protein expression is extended (the latter reflecting the experimental conditions as described in the original protocol; cf. Ref. 30). Thus, the magnitude of the insulin response is a function of the total amount of GLUT4 present in the cells, suggesting a saturation of the GLUT4 sorting and trafficking system. Likewise, an increase in basal cell surface GLUT4 levels in muscle and adipose cells was also observed in transgenic mice overexpressing GLUT4 (41). However, it remains unclear which component(s) of the GLUT4 trafficking system is (are) saturated by an excess of cellular GLUT4. It could be speculated that at least two trafficking steps might be affected by an excess of recombinant glucose transporters: (i) a hypothetical retention mechanism, responsible for the relatively low cell surface levels of GLUT4 in the basal state, and/or (ii) a sorting mechanism, responsible for the segregation of GLUT4 in the insulin-sensitive compartment. In the first scenario, a disproportionally greater amount of GLUT4 would be directed toward the plasma membrane in the basal state, as the expression of GLUT4 increased. The latter scenario predicts a disproportionally smaller amount of GLUT4 on the cell surface in the insulin-stimulated state with increasing GLUT4 expression. However, the molecular mechanisms of GLUT4 sorting and trafficking are as yet too poorly understood to distinguish between these two possibilities.
A possible involvement of the GTPase dynamin in GLUT4 trafficking has
been studied by co-transfecting rat adipose cells with HA-GLUT4 and
either wild-type dynamin1 or a GTP binding-deficient dynamin
1
mutant (K44A). A plasmid ratio (HA-GLUT4:dynamin) of 1:3 to 1:9 was
used to ensure an efficient coupling of the co-transfected plasmids.
Expression of both low amounts (3.5 h of protein expression) and high
amounts (24 h of protein expression) of recombinant wild-type dynamin
decreases cell surface HA-GLUT4 in both the basal and insulin-stimulated states (Fig. 2). This suggests that the endogenous dynamin activity is rate-limiting for endocytosis in rat adipose cells.
It could be argued that the endogenous dynamin activity in transfected
cells is saturated by an excess of recombinant GLUT4, leading to an
increased HA-GLUT4 internalization in wild-type dynamin-transfected
cells. Although we cannot exclude this possibility, it appears rather
unlikely since 3.5 h of protein expression using 0.5 µg of
plasmid/cuvette led to only a <2-fold increase in total cellular GLUT4
level/transfected cell as judged by Western blots using an anti-GLUT4
antibody and correcting for transfection efficiency (data not
shown).
In contrast, high levels of expression of mutant dynamin led to a
dramatic increase in basal cell surface glucose transporters (Fig.
2B), whereas intermediate levels of expression led to
partial effects (Fig. 2A). Moreover, the absolute amount of
cell surface HA-GLUT4 was increased by about 30-40% compared with the
insulin-stimulated control. This latter increase is in accord with a
previous observation that insulin stimulation leads to the net steady
state translocation of only 50% that of the total cellular GLUT4 to
the plasma membrane, whereas
50% of the glucose transporters
remains inside the cell, even in the continuous presence of insulin (1,
3). Thus, high expression levels of the mutant dynamin lead to an
effective accumulation of glucose transporters on the plasma membrane
where most or all of the cellular HA-GLUT4 is present on the cell
surface even in the basal state. Measurements of the kinetics of
HA-GLUT4 endocytosis were not sufficiently sensitive to demonstrate
an increased endocytosis with overexpression of wild-type dynamin
1. However, expression of dynamin-K44A even at intermediate levels clearly
shows that this dynamin mutant effectively blocks internalization of the glucose transporters. These results provide strong evidence that
functional dynamin is required for the internalization of the
GLUT4.
During preparation of this manuscript, Omata et al. (42)
reported the effects of overexpression of dynamin1 wild type and a
dynamin
1-K44E mutant in Chinese hamster ovary cells co-transfected with insulin receptors and GLUT4. The subcellular distribution of GLUT4
was analyzed nonquantitatively by immunofluorescence microscopy.
Consistent with our findings, expression of dynamin
1-K44E leads to an
increase in cell surface GLUT4 compared with dynamin
1 wild type in
cells not exposed to insulin. Likewise, wortmannin fails to decrease
cell surface GLUT4 in insulin-stimulated K44E-transfected cells.
However, in contrast to the present study, dynamin wild type increases
the cell surface levels of GLUT4 in insulin-stimulated cells. Despite
this apparently inconsistent result, the authors did not investigate
this effect further nor did they offer any explanation. Hence, it is
difficult to interpret these data based on the information provided. So
far, no comparable effect of dynamin on the cell surface levels of any
membrane protein has been reported. Thus, we conclude that this
observed phenomenon appears more likely to represent an artifact
originating from this particular expression system than the
physiological role of dynamin in the cell. After all, it has yet to be
established that the minimal insulin effect on glucose transporter
subcellular trafficking in heterologous expression systems such as
Chinese hamster ovary cells is in any way related to the dramatic
effects of insulin on GLUT4 in insulin target cells.
Early morphological studies in brown adipose tissue have revealed the presence of GLUT4 in clathrin-coated vesicles as well as in noncoated vesicles (3, 4). A study in our laboratory does not show an extensive co-localization of GLUT4 with clathrin (5), but due to the dynamics of GLUT4 trafficking, a large fraction of GLUT4 would not be expected to be associated with clathrin-coated vesicles. On the other hand, recent reports present evidence for a clathrin-dependent step in the endocytosis of GLUT4 on the basis of biochemical methods. Potassium (K+) depletion, known to inhibit the formation of clathrin-coated vesicles, inhibits GLUT4 endocytosis in rat adipose cells (13). However, inhibition of endocytosis of the insulin receptor, thought to be internalized by a clathrin-mediated process (43-45) is not observed with K+ depletion, despite an apparently enhanced insulin sensitivity of GLUT4 translocation to the plasma membrane (13). These data suggest that the effects of K+ depletion cannot be attributed only to an inhibition of clathrin-coated pit formation.
GLUT4 glucose transporters are reported to co-purify with clathrin from
brefeldin A-treated 3T3-L1 adipocytes (14) using differential
centrifugation of a Triton X-100-insoluble cell fraction. Nevertheless,
VIP21-caveolin (46), the main constituent of caveolae (review in Ref.
47), is also isolated by its detergent insolubility (48). Caveolae are
highly abundant in rat adipose cells and 3T3-L1 adipocytes (49, 50),
and an involvement of caveolae in GLUT4 trafficking is still debated
(50-52). In addition, the study by Corvera and co-workers (14) reveals
a 40-80% decrease in glucose transport activity in both the basal and
insulin-stimulated states after brefeldin A treatment, but others (53,
54) report no effects at all. Several recent studies demonstrate that
expression of dominant-negative dynamin1 inhibits the internalization
of transferrin receptors, epidermal growth factor receptors, and
2-adrenergic receptors in a variety of cultured mammalian cells (17,
18, 22, 25). Since these receptors are known to be internalized by a
clathrin-mediated mechanism, it is possible that GLUT4 are also
internalized by a clathrin-dependent pathway. However,
it has also been reported that the dynamin
1 homologue shibire may participate in clathrin-independent endocytosis
in Drosophila melanogaster (55, 56). The precise roles of
the dynamins in coated pit function and endocytosis via
nonclathrin-coated vesicles are unknown. Thus, the sensitivity of GLUT4
endocytosis to a dominant-negative mutant dynamin
1 does not
explicitly favor either of the two pathways.
In view of the well established effect of wortmannin to block the insulin-stimulated exocytosis of GLUT4 (Figs. 3B and 5C), we were surprised to observe that addition of wortmannin to insulin-stimulated, dynamin-K44A-expressing adipose cells synthesizing HA-GLUT4 did not change the amount of cell surface glucose transporters (Fig. 3C). Likewise, wortmannin did not inhibit the increase over time in cell surface HA-GLUT4 in basal cells synthesizing only tagged glucose transporters (Fig. 5A). Apparently, the pathway involved in targeting the newly synthesized recombinant glucose transporters to the plasma membrane is insensitive to wortmannin and thus different from the pathway involved in the insulin-induced translocation of GLUT4 to the cell surface. Two distinct post-Golgi biosynthetic pathways have been proposed for the delivery of newly synthesized membrane proteins to their endosomal/lysosomal compartments: from the TGN directly and indirectly by way of the plasma membrane. Major late endosomal and lysosomal membrane proteins (Lgps/Lamps) are transported from the TGN directly to endosomes and lysosomes (57). Asialoglycoprotein H1 and transferrin receptors traverse endosomes on their way from the TGN to the cell surface (58, 59), whereas the major histocompatibility complex class II molecules enter endosomes via the cell surface in HeLa cells (60). In analogy to the latter pathway, our findings suggest a biosynthetic route of GLUT4 where the glucose transporters are first directed from the TGN to the plasma membrane and then internalized by a dynamin-dependent pathway. Subsequently, GLUT4 are sorted into their intracellular compartment and thereby enter the insulin-sensitive GLUT4 recycling system. Newly synthesized GLUT4 leaving the TGN bypass the insulin-sensitive compartment and are targeted to the cell surface by means of a phosphatidylinositol 3-kinase-independent pathway, making the appearance of newly synthesized GLUT4 on the cell surface insensitive to wortmannin. However, once GLUT4 enters the insulin-sensitive compartment, their targeting to the plasma membrane becomes sensitive to wortmannin.
Taken together, the data show that the GTPase dynamin plays an important functional role in the endocytic portion of the GLUT4 trafficking pathway. Further work is needed to identify the specific components allowing dynamin to interact with the machinery required for GLUT4 internalization.
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ACKNOWLEDGEMENTS |
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We thank Drs. Jenny E. Hinshaw and Ian A. Simpson for helpful discussions and for critically reading the manuscript and Steven R. Richards and Mary Jane Zarnowski for expert technical assistance.
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FOOTNOTES |
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* This work was supported in part by a fellowship from the Deutsche Forschungsgemeinschaft (to H. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: EDMNS, DB, NIDDK,
National Institutes of Health, Bldg. 10, Rm. 5N102, 10 Center Dr. MSC
1420, Bethesda, MD 20892-1420. Tel.: 301-496-5953; Fax: 301-402-0432;
E-mail: hadi{at}helix.nih.gov.
1 The abbreviations used are: HA, hemagglutinin; TGN, trans-Golgi network.
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REFERENCES |
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