From the Division of Hematology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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The effect of wortmannin on the trafficking of
the mannose 6-phosphate/insulin-like growth factor II receptor
(Man-6-P/IGF-II receptor) and its ligand -glucuronidase has been
determined in murine L cells and normal rat kidney cells. The drug
induced a 90% decrease in the steady-state level of the Man-6-P/IGF-II
receptor at the plasma membrane without affecting the rate of
internalization, indicating that the return of receptor from endosomes
to the plasma membrane is retarded. Wortmannin also slowed the movement
of receptor from endosomes to the trans-Golgi network by
about 60%. Such a kinetic block would dramatically reduce the number
of Man-6-P/IGF-II receptors in the trans-Golgi network,
which could account for the previously described hypersecretion of
procathepsin D induced by wortmannin. In addition, the drug slowed
delivery of endocytosed
-glucuronidase from endosomes to dense
lysosomes. These data, taken together with the published reports of
others, indicate that wortmannin inhibits membrane trafficking out of
multiple compartments of the endosomal system and suggest a role for
phosphatidylinositol 3-kinase in regulating these processes.
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INTRODUCTION |
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The mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF-II receptor)1 follows a complex itinerary as it carries out its functions of transporting newly synthesized acid hydrolases to lysosomes and of internalizing ligands at the plasma membrane (1-4). In the trans-Golgi network (TGN), the receptor binds acid hydrolases and the complex is packaged into clathrin-coated vesicles. After budding from the TGN, the vesicles deliver their contents to early endosomes, and perhaps later endosomal compartments as well (5, 6). When the receptor with its bound ligand arrives in the late endosome, the acidic pH in the lumen of this compartment promotes ligand release. The free receptor recycles to the TGN to mediate another round of sorting, and the ligand travels to dense lysosomes (7-10). Cell surface Man-6-P/IGF-II receptors bind IGF-II in addition to extracellular acid hydrolases and are internalized via clathrin-coated vesicles. The endocytic and biosynthetic routes converge at the early endosome, and both pools of receptors mix freely (11-13). At steady state, the majority of the Man-6-P/IGF-II receptor is localized to endosomes with small amounts in the TGN and at the cell surface (8, 10, 14). The source of the cell surface receptor is not fully understood, but the majority may be transported from late endosomes (15).
While the role of clathrin-coated vesicles in the transport of the
receptor from the TGN and the plasma membrane to endosomal compartments
is well established, the nature of the vesicular carriers that mediate
subsequent steps of receptor trafficking is unknown. Recently, several
reports have documented that wortmannin, a fungal metabolite known to
inhibit phosphatidylinositol 3-kinases (PI 3-kinases), induces the
hypersecretion of the acid hydrolase procathepsin D (16, 17). Two
hypotheses have been suggested to explain this defect in lysosomal
enzyme sorting. One proposes that a wortmannin-sensitive enzyme is
required at the TGN either for the incorporation of Man-6-P/IGF-II
receptors into clathrin-coated vesicles or the formation of these
vesicles (16, 17). The second hypothesis suggests that such an enzyme
is required for recycling of the Man-6-P/IGF-II receptor from late
endosomes to the TGN and depletion of receptors from the TGN results in
procathepsin D secretion (18). It is clear from the initial studies
that wortmannin produced an altered cellular morphology, namely the appearance of swollen late endosomes containing Man-6-P/IGF-II receptors. This was associated with a loss of receptors from the TGN
and the cell surface (16, 18). These findings indicate that wortmannin
alters the trafficking of the Man-6-P/IGF-II receptor and suggests a
role for PI 3-kinases in this process. However, since these studies
primarily utilized morphologic techniques to evaluate receptor
distribution, it was not possible to assess the effect of wortmannin on
the actual kinetics of receptor transport between compartments.
Furthermore, recent work has reported that this altered endosomal
morphology does not reflect a block in receptor transport to the TGN
(19). To address this issue directly, we have analyzed the effect of
wortmannin on the trafficking of the Man-6-P/IGF-II receptor using a
variety of kinetic assays in living cells. In addition, we have
analyzed the effect of this fungal metabolite on the transport of
-glucuronidase from the cell surface to dense lysosomes. The results
of these experiments indicate that wortmannin retards the movement of
both the Man-6-P/IGF-II receptor and
-glucuronidase out of late
endosomes.
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EXPERIMENTAL PROCEDURES |
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Materials--
All reagents were of analytical grade.
Na125I was obtained from Amersham Corp.;
UDP-[6-3H]galactose was from American Radiolabeled
Chemicals (St. Louis, MO); lactoperoxidase was from Calbiochem (San
Diego, CA); Percoll was from Pharmacia (Uppsala, Sweden);
galactosyltransferase was from Fluka (Buchs, Switzerland). All other
enzymes and chemicals were obtained from Sigma. Wortmannin was prepared
as a 2 mM stock in Me2SO and stored at
20 °C. Aliquots were thawed only once, diluted, and used
immediately.
Cells--
Mouse L cells stably expressing the bovine
Man-6-P/IGF-II receptor (Cc2 cells) or Man-6-P/IGF-II receptor with a
truncated cytoplasmic tail (344 cells) have been described previously
(20, 21). The mutant receptor expressed by 344 cells has an Ala
substitution at Tyr24, and the cytoplasmic tail is
truncated to 29 amino acids. Cell lines were grown in complete medium
(-minimal essential medium containing 10% heat-inactivated fetal
calf serum, 100 units/ml penicillin G, 100 µg/ml streptomycin
sulfate) with 350 µg/ml G418 (Life Technologies, Inc.) at 37 °C in
5% CO2. Normal rat kidney cells were obtained from ATCC
and grown in complete medium.
Endocytosis of -Glucuronidase--
-Glucuronidase was
purified from the secretions of 13.2.1 cells (a gift from Dr. W. Sly,
St. Louis University) as described previously (20). Cc2 cells were
seeded in 22-mm wells of a 12-well tissue culture plate and grown to
confluence, washed with PBS, and incubated with 10 nM
-glucuronidase in complete medium in the absence or presence of 1 µM wortmannin for appropriate times. Medium was
aspirated, and cells were washed with PBS five times, washed with 15 mM phosphate-citrate buffer (pH 5.0) twice, and lysed with
PBS+1% Triton X-100. An aliquot of the lysate was assayed for
-glucuronidase activity.
Iodination of -Glucuronidase--
-Glucuronidase (30 µg)
was iodinated with 1 mCi of Na125I using soluble
lactoperoxidase as described (22) and gel-filtered (20). The specific
radioactivity of the pooled
-glucuronidase was 8-16 µCi/µg,
assuming complete recovery of the
-glucuronidase.
Assay for Rapid Receptor Internalization--
The short
internalization assay was performed as described previously (20) with
minor modifications as described below. Briefly, 344 or Cc2 cells were
seeded into 22-mm wells of a 12-well plate and grown to confluence.
When determining the internalization rate in the presence of
wortmannin, the cells were pretreated with complete medium containing 1 µM wortmannin for 30 min. The cells were rinsed with
complete medium, and then 1 ml of complete medium containing 0.1 µg
(approximately 1 × 106 cpm) of
125I--glucuronidase in the absence or presence of 1 µM wortmannin was added. After a 30-min incubation on
ice, the cells were rapidly washed five times with ice-cold PBS
containing 1% bovine serum albumin and the plate was transferred to a
37 °C water bath. A 0.5-ml portion of a mixture of 0.035% trypsin,
0.013% EDTA, 10 mM Man-6-P in 15 mM
phosphate-citrate saline, pH 5, was immediately added to each of two
wells that were used for measurement of the total surface binding of
ligand, while the other wells received an addition of 0.5 ml of
37 °C complete medium containing 1 µM wortmannin when
appropriate. After the incubation period, the medium was collected and
0.5 ml of the pH 5 trypsin/Man-6-P mixture was added to each well. At
the end of an additional 3-min incubation at 37 °C, 0.8 ml of
complete medium was added to each well. The cells were then harvested,
pelleted, and the supernatant aspirated. The radioactivity in the cell
pellet was measured in a
-counter. The cells from the two wells used
to determine the total surface binding were treated similarly except
that the radioactivity of the harvesting medium was measured directly,
prior to pelleting the cells.
Cell Surface Binding of
125I--Glucuronidase--
Cc2 cells were seeded in a
24-well tissue culture plate and grown to confluence. Cells were washed
with complete medium and then incubated with 0.5 ml of complete medium
containing 1 µM wortmannin for up to 3 h. The
wortmannin-containing medium was replaced hourly. Cells were then
subjected to the same procedure described for determining total surface
binding in the rapid internalization assay.
Percoll Gradient Fractionation--
Percoll gradient
fractionation was performed as described previously (23) with minor
modifications as described below. Briefly, confluent cultures of normal
rat kidney cells in 100-mm Petri dishes were preincubated or not with 1 µM wortmannin and then incubated with 3 ml of complete
medium containing 2 × 106 cpm of
125I--glucuronidase in the absence or presence of 1 µM wortmannin at 37 °C for an additional 15 min. The
cells were washed twice with PBS and incubated with 10 ml of complete
medium for up to 90 min in the absence or presence of 1 µM wortmannin. The wortmannin-containing medium was
replaced hourly. After two washes with PBS, cells were scraped into 2 ml of homogenization buffer (HB) (0.25 M sucrose, 1 mM EDTA, pH 7.5) and pelleted for 10 min at 140 × g at 4 °C. The cells were resuspended in 850 µl of HB,
and passed 12 times through a ball bearing homogenizer (24) with a
clearance of 51.2 µm. The homogenate was diluted to 1.7 ml and
centrifuged, and the resulting postnuclear supernatant was layered over
a discontinuous gradient consisting of a 1.2-ml cushion of 10 × HB and 8.5 ml of an 18% Percoll solution in 1 × HB. The gradient
was centrifuged for 30 min at 20,000 rpm in a Ti 50 rotor (Beckman
Instruments, Palo Alto, CA) at 4 °C. The gradient was collected from
the bottom of the tube in 1-ml fractions. The amount of radioactivity
in each fraction was measured in a
-counter, and an aliquot of each fraction was assayed for
-hexosaminidase activity after the sample was adjusted to 1% Triton X-100 and incubated on ice for 1 h.
Enzyme Assays--
-Glucuronidase activity was determined by
dilution of the samples in 100 mM 4-methlyumbelliferyl
-D-glucuronide, 100 mM sodium acetate, pH
4.8. The samples were incubated for 15-60 min at 37 °C; the
reaction was stopped with the addition of 0.25 M glycine,
pH 10.5, and the fluorescence was measured.
Sialylation Assay--
The assay was performed as described
previously (12) with a few modifications (25). Briefly, mouse L cells
were incubated in complete medium containing 0.02 unit/ml Vibrio
cholerae neuraminidase and 0.02 unit/ml Diplococcus
pneumoniae -galactosidase for 1 h at 37 °C. The
glycosidases were removed by washing cells three times with PBS, and 10 µM 2,3-dehydro-2-desoxy-N-acetylneuraminic acid was added to all subsequent incubations to abolish the activity of
any neuraminidase that may have been internalized by pinocytosis. Cells
were incubated for 30 min with 20 µCi/ml
UDP-[6-3H]galactose and 0.2 unit/ml galactosyltransferase
on ice to label surface glycoproteins, washed, and then incubated at
37 °C for 2 h in the absence or presence of 1 µM
wortmannin. The wortmannin-containing medium was changed after the
first hour. The cells were collected by scraping, washed with PBS, and
sonicated in lysis buffer (50 mM imidazole HCl, pH 6.5, 150 mM NaCl, 5 mM EDTA, 2% Triton X-100, 0.5%
deoxycholate, and 2 µg/ml each of pepstatin A, leupeptin, chymostatin, and antipain, and 10 trypsin inhibitory units/ml of
aprotinin), followed by centrifugation at 50,000 × g
for 30 min at 4 °C. The supernatant containing the solubilized
Man-6-P/IGF-II receptor was applied to a phosphopentamannose-Sepharose
affinity column. The column was washed with G-25 buffer (50 mM imidazole, pH 6.5, 150 mM NaCl, 0.25%
Triton X-100, 5 mM EDTA, 0.5 mg/ml bovine serum albumin,
and protease inhibitors) and the bound Man-6-P/IGF-II receptor was
eluted with G-25 buffer containing 10 mM Man-6-P. The
Man-6-P eluted fractions were pooled and digested for 24 h at
37 °C with 0.02 unit/ml D. pneumoniae
-galactosidase.
This material was applied to a Sephadex G-25 column, and the
radioactivity in the void volume (Man-6-P/IGF-II receptor) and in the
included peak (free [3H]galactose) was determined. The
percent sialylation of the Man-6-P/IGF-II receptor was calculated by
dividing the radioactivity recovered in the void volume by the sum of
the radioactivity in the void and included fractions, and multiplying
by 100.
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RESULTS |
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Wortmannin Decreases the Uptake of -Glucuronidase by Mouse L
Cells--
As the first step toward investigating the effects of
wortmannin on the trafficking of the Man-6-P/IGF-II receptor, we
measured the endocytosis of the acid hydrolase
-glucuronidase by
mouse L cells in the presence of this fungal metabolite. The uptake of
this enzyme is known to be mediated by the Man-6-P/IGF-II receptor (26,
27). Mouse L cells stably expressing the bovine Man-6-P/IGF-II receptor
(Cc2 cells) were incubated with
-glucuronidase for 1 h in the
absence or presence of increasing concentrations of wortmannin (10 nM to 10 µM). As shown in Fig.
1, inhibition of
-glucuronidase uptake
was detected at a wortmannin concentration of 10 nM and half-maximal inhibition occurred at 40 nM. The maximal
inhibition was 91%.
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Wortmannin Induces a Decrease in Cell Surface Man-6-P/IGF-II
Receptors--
The inhibition of -glucuronidase uptake in the
presence of wortmannin could be due to a loss of cell surface receptors
or a decrease in their rate of internalization. To determine if
the number of cell surface Man-6-P/IGF-II receptors was diminished, Cc2
cells were treated with 1 µM wortmannin for up to 3 h at 37 °C and then the surface binding of
125I-
-glucuronidase was measured on ice. As shown
in Fig. 4, wortmannin caused a rapid
decline in the amount of 125I-
-glucuronidase that bound
to the cell surface receptors. The maximal decrease of 90% was reached
with a t1/2 of approximately 15 min.
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Wortmannin Does Not Alter the Rate of Internalization of the
Man-6-P/IGF-II Receptor--
The effect of wortmannin on the initial
rate of receptor internalization was determined as follows;
125I--glucuronidase was allowed to bind on ice to cells
that had been pretreated or not for 30 min with 1 µM
wortmannin. The unbound ligand was removed, and the cells were warmed
to 37 °C. Receptor internalization was followed by determining the
rate of uptake of
-glucuronidase over a 2-min period in the absence
or presence of wortmannin. This experiment was initially performed with
L cells expressing the wild-type receptor, but the binding of
125I-
-glucuronidase to the cells preincubated with
wortmannin was reduced so much that valid measurements could not be
obtained (Fig. 4). To overcome this technical problem, the 344 cells
were used because they have more surface receptors than Cc2 cells. The
Man-6-P/IGF-II receptor expressed in 344 cells has a truncated (29-amino acid) tail, but it is internalized at the same rate as the
wild-type receptor (20). Furthermore, the truncated receptor is lost
from the cell surface of wortmannin-treated 344 cells with kinetics
identical to those described for the full-length receptor in the Cc2
cell line (data not shown). Fig. 5 shows
that the presence of 1 µM wortmannin had no effect on the
initial rate of
-glucuronidase internalization. Since wortmannin
causes the number of cell surface receptors to decrease without
altering the rate of receptor internalization, the return of receptors to the cell surface must be inhibited.
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Wortmannin Slows the Return of the Man-6-P/IGF-II Receptor to the
TGN--
One explanation for the defective acid hydrolase sorting
induced by wortmannin is that recycling of the Man-6-P/IGF-II receptor to the TGN is impaired. To measure this step, we took advantage of the
fact that the receptor is a glycoprotein that can be acted upon by
sialyltransferases localized in the TGN (28-30). Consequently, the
actions of these glycosyltransferases can be used to mark the return of
receptor molecules to the Golgi. However, the Asn-linked oligosaccharides on the receptor must express a terminal galactose to
be substrates for the sialyltransferases. Therefore, we pretreated mouse L cells with neuraminidase and -galactosidase to remove the
sialic acid and galactose residues of the cellular glycoproteins. The
resulting terminal N-acetylglucosamine residues of the cell surface glycoproteins (including the Man-6-P/IGF-II receptor) were
labeled with [3H]galactose by incubating the cells with
exogenous galactosyltransferase and UDP-[6-3H]galactose
on ice. The cells were then warmed to 37 °C in the absence or
presence of wortmannin for 2 h to allow the surface-labeled receptor molecules to be internalized and return to the TGN, where sialic acid residues are transferred on to the
[3H]galactose by sialyltransferase. To determine the rate
of sialylation, the receptor was isolated on a
phosphopentamannose-Sepharose affinity column and treated with
-galactosidase, which releases terminal [3H]galactose
residues, but not [3H]galactose residues that have been
modified through the addition of sialic acid moieties. The released
[3H]galactose was then separated from the receptor by gel
filtration. The fraction of the [3H]galactose that is
resistant to
-galactosidase reflects the rate of return of receptor
molecules to the Golgi and the efficiency of their sialylation.
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Wortmannin Slows the Arrival of Endocytosed -Glucuronidase in
Dense Lysosomes--
After the Man-6-P/IGF-II receptor with its bound
ligand is internalized at the plasma membrane, the complex is
transported through early endosomes to a late endosomal compartment,
where the acidic pH promotes the release of the ligand, which is
subsequently delivered to the dense lysosome. The studies described in
the previous section show that wortmannin does not affect the initial rate of internalization of
-glucuronidase, which is mediated by the
Man-6-P/IGF-II receptor. To determine if this fungal metabolite influences the later steps in this pathway, we followed the delivery of
125I-
-glucuronidase to dense lysosomes in the presence
and absence of this agent. Control and wortmannin pretreated normal rat
kidney cells were allowed to take up 125I-
-glucuronidase
for 15 min. Following chase times of up to 90 min, the cells were
homogenized and the postnuclear supernatants were fractionated on 18%
Percoll gradients to separate dense lysosomes from other membranous
compartments (including endosomes) that were recovered near the top of
the gradient. Fractions were collected and the radioactivity in each
fraction determined, as shown in Fig. 6.
To identify the position of dense lysosomes in the gradient, each
fraction was assayed for the lysosomal marker
-hexosaminidase.
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DISCUSSION |
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The data presented in this paper demonstrate that wortmannin slows the movement of the Man-6-P/IGF-II receptor from endosomes to the TGN and the plasma membrane without affecting the rate of internalization at the plasma membrane. These changes in the kinetics of receptor trafficking would be expected to alter the steady-state distribution of the Man-6-P/IGF-II receptor such that the level at the plasma membrane and TGN would decrease, whereas it increases in endosomes. This, in fact, is what has been observed in morphologic studies examining the effect of wortmannin on the distribution of the Man-6-P/IGF-II receptor (16, 18). These investigators found that, within 30 min, wortmannin caused the receptor to accumulate in swollen late endosomes while being depleted from the TGN and plasma membrane. In our experiments, wortmannin treatment of murine L cells resulted in a 90% reduction in the level of Man-6-P/IGF-II receptor at the cell surface and slowed the return of receptor to the TGN by 60%.
These kinetic effects on the trafficking of the Man-6-P/IGF-II receptor help to explain the hypersecretion of procathepsin D that occurs in response to this agent (16, 17). By slowing the return of the Man-6-P/IGF-II receptor to the TGN, wortmannin treatment would reduce the number of receptor molecules in that compartment available for binding newly synthesized acid hydrolases, including procathepsin D. When the number of available receptor molecules becomes insufficient to bind the newly synthesized acid hydrolases, hypersecretion will result. Indeed, Gaffet et al. (31) have shown recently that clathrin-coated vesicles isolated from wortmannin-treated cells contain significantly less Man-6-P/IGF-II receptor. This finding is consistent with a loss of receptor from the TGN. Furthermore, this work demonstrates that wortmannin does not prevent the formation or consumption of TGN derived clathrin-coated vesicles. While this manuscript was in preparation, Nakajima et al. (19) reported that wortmannin does not effect recycling of the Man-6-P/IGF-II receptor from late endosomes to the TGN in Chinese hamster ovary and K562 cells. While the reason for this disparity is not clear, it may reflect differences in the cell types studied or differences in the nature of the assay systems used, such as the use of lectin chromatography to determine the fraction of receptors bearing sialic acid residues.
Wortmannin also caused a marked delay in the arrival of endocytosed
-glucuronidase in dense lysosomes. Since wortmannin does not
dissipate the endosomal pH gradient, this delay cannot be attributed to
the absence of the pH-induced release of ligand from the Man-6-P/IGF-II
receptor that occurs in the late endosome (16, 32). Rather, these
findings provide further support for a general inhibitory effect of
wortmannin on the movement of proteins out of endosomes, in this case
resulting in slowed delivery to lysosomes. Since the initial processing
of procathepsin D occurs in late endosomes while the final cleavage
that yields the mature form requires delivery of the acid hydrolase to
dense lysosomes, wortmannin treatment would be expected to slow the
production of mature cathepsin D. Indeed, previous work that monitored
the effect of this agent on the processing of retained procathepsin D
indicates that the conversion of the intermediate form of cathepsin D
to the mature form is impaired in wortmannin-treated cells, lending
further evidence for reduced trafficking from late endosomes to
lysosomes (17). Another study following the degradation of endocytosed
Semliki Forest virus as an indicator of lysosomal delivery also found
that wortmannin delays the breakdown of internalized virus (32).
Although these studies demonstrate that movement of proteins from
endosomes to lysosomes is slowed by wortmannin, we cannot determine
whether the delivery is delayed because of a direct inhibition of
fusion of late endosomal multivesicular bodies with lysosomes as
described for the epidermal growth factor receptor (33) or if the
formation of vesicular carriers is impaired.
It is of interest that wortmannin has recently been shown to cause a
redistribution of lysosomal membrane proteins from dense lysosomes to
swollen late endosomes distinct from those containing the
Man-6-P/IGF-II receptor (18). This suggests that retrograde trafficking
from dense lysosomes proceeds in the presence of this drug while
movement of proteins from late endosomes to lysosomes is inhibited. A
similar phenomenon may be occurring in our experiments, which would
account for the shift of -hexosaminidase from dense lysosomes to
lighter fractions after prolonged wortmannin treatment (Fig. 6). The
lysosomal marker could also localize at the top of the gradient if the
depletion of lysosomal membrane proteins from lysosomes made these
organelles more susceptible to lysis during homogenization. However,
the activity recovered was latent without detergent treatment,
consistent with the
-hexosaminidase being within vesicles (data not
shown). Furthermore, it has been shown that
-galactosidase and Lamp
1 redistribute from lysosomes to early endosomes when transport out of
early endosomes is blocked in a mutant cell line
(34).2 Such observations
suggest that some soluble lysosomal enzymes, in addition to lysosomal
membrane proteins, may continually cycle between lysosomes and
endosomes.
Although the results of this study implicate the late endosome as a target of wortmannin action, this agent has been shown to act at additional sites within the endosomal system. For example, transferrin receptor recycling, which involves early endosomal compartments, is inhibited by wortmannin while endocytosis of this receptor has been reported to be either increased or decreased (32, 35-38). In in vitro assays, the drug inhibited early endosome fusion (36, 37, 39). Wortmannin has also been reported to inhibit ligand-induced down-regulation of the platelet-derived growth factor receptor (35, 40, 41) and transcytosis in polarized epithelial cells (42). It also inhibits the insulin-induced exocytosis of the GLUT4 transporter (43-45) and IGF-II-stimulated surface expression of the Man-6-P/IGF-II receptor (46). These effects are elicited by low nanomolar concentrations of the drug, and the p110 PI 3-kinase has been implicated as a mediator of these effects. On the other hand, micromolar concentrations of wortmannin were required to induce lysosomal enzyme hypersecretion in Chinese hamster ovary cells, suggesting that one or more enzymes with differential wortmannin sensitivity may be involved in producing this effect (32). Therefore, in comparing the concentration of wortmannin required to achieve an effect, one must bear in mind that the drug is metabolized and that the rate of metabolism may vary among cell types.
As pointed out by Reaves et al. (18), the common theme of
these effects of wortmannin is inhibition of membrane traffic out of
the endosomal system with traffic into this system from the plasma
membrane, TGN, and lysosome being relatively unaffected or possibly not
affected at all as a primary event. Our data on the trafficking of the
Man-6-P/IGF-II receptor and endocytosed -glucuronidase are
consistent with this notion and further demonstrate that wortmannin
slows traffic out of endosomes rather than causing a complete
block.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. F. Murphy for sharing unpublished data from his laboratory with us. We are grateful to Dr. W. Sly for the generous gift of the 13.2.1 cells and to Dr. Ian Trowbridge for providing the BW 5147 PHAR1.8 cells. We thank Walter Gregory and Carolyn Noll for excellent technical assistance and members of the Kornfeld and Majerus laboratories for helpful discussions and critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA 08759-30 (to S. K.), NRSA Grant for Training in Molecular Hematology T32 HL07088 (to R. K.), and MSTP Training Grant T32 GM07200 (to R. K.)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: Div. of Hematology,
Washington University School of Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110. Tel.: 314-362-8803; Fax: 314-362-8826; E-mail:
skornfel{at}im.wustl.edu.
1 The abbreviations used are: Man-6-P/IGF-II receptor, mannose 6-phosphate/insulin-like growth factor II receptor; Man-6-P, mannose 6-phosphate; IGF-II, insulin-like growth factor II; HB, homogenization buffer; PI 3-kinase, phosphatidylinositol 3-kinase; TGN, trans-Golgi network; PBS, phosphate-buffered saline.
2 J. Wightman, J. A. Schwartz, and R. F. Murphy, personal communication.
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REFERENCES |
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