From the
We have previously reported that platelet-derived growth factor
(PDGF) receptor mutants that lack high affinity binding sites for
phosphatidylinositol 3-kinase (PI 3-kinase) fail to concentrate in
juxtanuclear vesicular structures after activation with PDGF. We have
now identified the point in the endocytic pathway at which PI 3-kinase
binding sites are required. Receptor internalization from the plasma
membrane, measured as the acquisition of acid resistance of prebound
Stimulation of receptor tyrosine kinases rapidly elicits a large
number of intracellular events including actin filament reorganization,
membrane ruffling, transmembrane ion fluxes, and changes in the
activity of metabolic enzymes. One of the earliest responses to
stimulation is the internalization of the activated receptor.
Internalized receptors can recycle back to the cell surface
(1) ,
or can be sorted to the lysosome for degradation. This latter process
results in the down-regulation of cell surface receptors. Receptor
down-regulation appears to be an important element in the control of
cellular proliferation, since mutations that impair the ability of some
receptors to internalize and degrade cause cell
transformation
(2) .
In addition to its physiological
relevance, the down-regulation of receptor tyrosine kinases offers a
useful model system to study the molecular events involved in the
trafficking of proteins from the plasma membrane to the lysosome,
because receptor movement can be selectively and synchronously elicited
by addition of specific growth factors
(24) . Internalization of
receptor tyrosine kinases often involves both high affinity, low
capacity and low affinity, high capacity pathways. The high affinity,
low capacity internalization depends on tyrosine phosphorylation of the
receptor cytoplasmic domain (3-7). Following internalization,
some receptor tyrosine kinases are directed to the lysosomal pathway.
The molecular requirements involved in this sorting process are not
understood.
We have recently explored the hypothesis that the SH2
domain-containing proteins that are recruited to receptor cytoplasmic
domains after activation may play a role in the trafficking of receptor
tyrosine kinases
(8, 9) . Tyrosine autophosphorylation of
the PDGF
These results raise important questions regarding
the point in the endocytic pathway at which PI 3-kinase binding sites
are required and the biochemical basis for this requirement. The
results shown here strongly suggest that PI 3-kinase binding sites are
required at a postendocytic step, where the sorting of activated PDGF
receptors to the lysosome occurs. Furthermore, wortmannin, which has
recently been shown to be a potent (IC
Cells
were incubated with tracer concentrations (2 ng/ml) of
A pronounced decrease in the
levels of labeled wild-type and Y40/51 receptors was observed in these
experiments (Fig. 5). In contrast, neither F5 mutant receptors
nor the other add-back mutant receptors were appreciably degraded after
a similar time of exposure to ligand. These results support the
hypothesis that intact PI 3-kinase binding sites are required for the
trafficking of activated PDGF receptors to the lysosomal pathway.
Receptors that rapidly concentrate in juxtanuclear vesicular structures
(9) after exposure to PDGF are efficiently degraded, consistent
with these structures corresponding to late endosomes, multivesicular
bodies, and lysosomes.
We first analyzed the effects of
wortmannin on receptor down-regulation from the cell surface
(Fig. 6). Cells expressing wild-type receptors, the F5 mutant,
and the Y40/51 add-back mutant were incubated without or with
wortmannin (50 nM) for 10 min and then exposed to PDGF (50
ng/ml) for 30-90 min at 37 °C. After a brief acid wash, the
binding of
We have previously reported that the trafficking of PDGF
receptors requires PI 3-kinase binding sites
(9) . A critical
question raised by this finding is at what point in the pathway of PDGF
receptor internalization are these sites required. Here we show that
the requirement for PI 3-kinase binding sites is not during the initial
steps of endocytosis. These results are consistent with the findings of
Mori et al.(25) who have shown that only mutations in
tyrosine 579 of the PDGF receptor cytoplasmic domain significantly
affect this process. The initial internalization of a receptor mutant
lacking tyrosines 740, 751, 771, 1009, and 1021 was decreased by 30%,
but restoration of any of the missing tyrosine residues was sufficient
to restore the wild-type internalization rates. These results indicate
that these residues play a small individual role in the internalization
of the PDGF receptor. However, the results also suggest that multiple
tyrosine residues in the receptor cytoplasmic domain can function as
weak internalization signals. The net rate of internalization is
probably the product of all these signals. PI 3-kinase binding sites
appear to be necessary at a step subsequent to internalization. Without
PI 3-kinase binding sites receptors escape degradation and appear to
recycle to the cell surface.
A second question addressed in this
study relates to the nature of the requirement for PI 3-kinase binding
sites. These sites are known to bind the p85 subunit of PI 3-kinase but
also the adaptor protein Nck (16) and possibly other unknown factors.
The p85 subunit of PI 3-kinase interacts with dynamin, a GTPase that is
critically required for endocytosis. Thus, the requirement for PI
3-kinase binding sites for PDGF receptor trafficking could reflect a
requirement for p85, for p110 catalytic activity, for Nck, or for a yet
unknown factor. The finding that wortmannin, a potent inhibitor of PI
3-kinase activity, blocks efficient down-regulation and degradation of
wild-type receptors suggests strongly that the requirement for PI
3-kinase binding sites reflects a requirement for PI 3-kinase activity.
A role for PI 3-kinase activity in sorting of membrane proteins has
been clearly established in yeast, where the only known PI 3-kinase,
vps34, is required for the sorting of newly synthesized hydrolases to
the yeast vacuole
(26) . Interestingly, vps34 has also been
recently discovered in a screen for endocytosis-dependent growth, which
monitors endocytosis from the cell surface to the vacuole
(27) .
Experiments on these mutants have shown that vps34/end12 is not
necessary for internalization of
A requirement for PI 3-kinase in steps
subsequent to initial internalization suggests that postendocytic
receptor sorting requires active receptor tyrosine kinase activity in
order to maintain an association with the SH2 domains of p85. It has
been shown that the internalized EGF and PDGF receptors have an active
tyrosine kinase activity
(8, 28) and that the activity
of the EGF receptor kinase is necessary for sorting the EGF receptor to
the lysosome
(29) . This view, however, has been questioned
recently in experiments that conclude that postendocytic trafficking of
EGF receptors is independent of their intrinsic tyrosine kinase
activity. In this latter paper however, degradation of EGF ligand, not
of EGF receptor, was analyzed
(30) .
An important question
raised by these results is whether PI 3-kinase binding is required for
the down-regulation of other receptor tyrosine kinases in mammalian
cells. In this regard, it is interesting that the insulin receptor,
which escapes degradation after internalization
(1) , fails to
bind PI 3-kinase directly
(31) . The postendocytic destiny of the
EGF receptor varies. In some cells internalized receptors down-regulate
efficiently
(29, 30) , whereas in others they recycle to
the cell surface
(32, 33) . Interestingly, the EGF
receptor binds PI 3-kinase poorly, with a 12-fold lower affinity than
the PDGF receptor (34). However, in some cells EGF receptors dimerize
with ErbB3, which binds PI 3-kinase with an affinity comparable with
the PDGF receptor (35). It is interesting to speculate on the
possibility that the observed differences in the ability of EGF
receptors to efficiently down-regulate may correlate with their ability
to dimerize with ErbB3 and thus interact strongly with PI 3-kinase. The
availability of wortmannin will be useful in determining whether the
requirement for PI 3-kinase for receptor down-regulation is universal.
PI 3-kinase in mammalian cells has been implicated in several
functions of receptor tyrosine kinases, including mitogenic signaling,
actin filament organization, chemotaxis, and
down-regulation
(36) . The molecular mechanisms whereby PI
3-kinase mediates these diverse cellular functions are unknown. It is
possible that each of the lipid products of mammalian PI 3-kinase,
which include PI-3-P, PI-3,4-P
Immunofluorescence studies were done using
instrumentation of the Biomedical Imaging Group at the University of
Massachusetts. We thank David Hartley and Howard Shpetner for helpful
discussions and critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-PDGF, was only slightly decreased in cells expressing a
PDGF receptor mutant (F5) lacking PI 3-kinase, GTPase-activating
protein (GAP), phospholipase C
, and Syp binding sites but not
expressing mutants where any of these individual sites were restored
nor expressing a mutant lacking exclusively PI 3-kinase binding sites.
In contrast, the extent of down-regulation of PDGF binding sites from
the cell surface after prolonged incubation with PDGF as well as the
degradation of [
S]methionine-labeled receptor
were markedly reduced in cells expressing the F5 mutant, mutants
restored in GAP, phospholipase C
, or Syp binding sites or
expressing the mutant exclusively lacking PI 3-kinase binding sites but
not in cells expressing the mutant where PI 3-kinase binding sites were
restored. Inhibition of PI 3-kinase activity with wortmannin caused a
dramatic decrease in the rates of down-regulation and degradation of
wild-type receptors. These results suggest that PI 3-kinase binding
sites are not required for internalization of PDGF receptor but are
required to divert the PDGF receptor to a degradative pathway.
Furthermore, the requirement for PI 3-kinase binding sites on the
receptor appears to be due to a requirement for PI 3-kinase catalytic
activity.
(
)
receptor creates high affinity binding
sites for several proteins including phosphatidylinositol 3-kinase (PI
3-kinase), phospholipase C
, a GTPase-activating protein for Ras
(GAP), a 65-kDa phosphotyrosine phosphatase (Syp), non-receptor
tyrosine kinases, and the adaptor proteins Grb2 and
Nck
(10, 11, 12, 13, 14, 15, 16) .
A PDGF receptor mutant that can autophosphorylate but lacks the major
high affinity binding sites for PI 3-kinase, GAP, phospholipase C
,
and Syp displays striking alterations in cellular trafficking patterns
(9, 17); whereas wild-type receptors concentrate in a juxtanuclear
vesicular compartment, the mutant remains dispersed at the cell
periphery
(9) . Restoration of the PI 3-kinase binding sites
completely restores the ability of receptors to concentrate
intracellularly.
< 10
nM) inhibitor of the p110 catalytic subunit of mammalian PI
3-kinase
(18, 19) , inhibits the down-regulation and
degradation of activated receptor, suggesting that the requirement for
PI 3-kinase binding sites reflects a requirement for PI 3-kinase
catalytic activity.
Cells and Constructs
The F5(
)
mutant is a receptor in which tyrosines 740, 751, 771,
1009, and 1021 were substituted with phenylalanine and is deficient in
the binding of PI 3-kinase, phospholipase C
, GAP and
Syp
(17) . Receptor constructs selectively restored in the high
affinity binding of PI 3-kinase (Y40/51), phospholipase C
(Y1021),
GAP (Y771), or Syp (Y1009) were generated by mutating the corresponding
phenylalanines in F5 back to tyrosine. In addition, a receptor
exclusively deficient in PI 3-kinase binding (F40/51) was constructed
by mutating Tyr
and Tyr
in the wild-type
receptor to phenylalanine. Each construct was introduced into human
HepG2 cells, which do not express endogenous
-PDGF receptors, with
the pLXSN retroviral expression vector
(17) . All constructs were
expressed at approximately 5
10
receptors/cell
(17) and were activated by ligand
(9, 17) . Cells
were grown to 80% confluence in Dulbecco's modified Eagle's
medium (DMEM) supplemented with fetal calf serum (10%) (UBI) containing
G418 (0.5 mg/ml).
Recombinant PDGF-BB was labeled with
I-PDGF
Internalization
I to 21,000 cpm/ng by the method of Bolton and Hunter
according to the manufacturer's instructions (Amersham Corp.).
Cells expressing wild-type or mutant PDGF receptors or the empty pLXSN
vector were grown in 12-well multiwell dishes to 75% confluence and
serum-starved for 18 h. PDGF internalization experiments were conducted
as described by Mori et al.(20) . Cells were washed
twice with ice-cold DMEM and incubated with 0.5 ml of DMEM containing 1
mg/ml bovine serum albumin and 50,000 cpm of
I-PDGF.
After 90 min of incubation at 5 °C, monolayers were washed three
times with ice-cold binding medium. Binding medium at 37 °C was
then added for 0-20 min, and cells were rapidly cooled by placing
the multiwell dishes on ice and washing with ice-cold binding medium.
Cells were then incubated for 5 min with 500 µl of
phosphate-buffered saline containing 1 mg/ml bovine serum albumin, pH
7.5, or with the same buffer at pH 3.5. This acid wash procedure has
been shown to release approximately 90% of cell surface bound
radioactivity
(20) . Cells were then washed three times with
binding medium, and monolayers were dissolved in a lysis buffer
composed of Triton X-100 (1%), glycerol (10%), and Tris, pH 7.5 (20
mM). Radioactivity associated with the acid-washed monolayers
(internalized) or the non-acid washed monolayers (total
cell-associated) was measured by
counting.
Receptor Down-regulation
Cells expressing
wild-type or mutant PDGF receptors or the empty pLXSN vector were
cultured and serum-starved as described above. Monolayers were
incubated with DMEM or DMEM containing 0-20 ng/ml PDGF-BB for
30-90 min at 37 °C. Cells were then cooled and acid-washed as
described above. After the acid wash, cells were washed three times
with ice-cold DMEM and incubated for 90 min at 5 °C with 0.5 ml of
DMEM containing 1 mg/ml bovine serum albumin and 100,000 cpm of
I-PDGF. Monolayers were washed three times and dissolved
in lysis buffer. Nonspecific binding was defined as the radioactivity
bound to monolayers of HepG2 cells transfected with pLXSN empty vector.
Receptor Degradation
Cells were grown to 85%
confluence in 60-mm culture dishes and incubated for 18 h in
serum-free, methionine-free DMEM containing
[S]methionine (100 µCi/ml) (Amersham Corp.)
and bovine serum albumin (1%) and then for 45 min in DMEM containing
bovine serum albumin (1%) and methionine (0.3 mg/ml) (DMEM-bovine serum
albumin). Cells were then incubated without or with PDGF-BB (50 ng/ml)
(UBI) for 2 h. Cells were washed twice in ice-cold phosphate-buffered
saline and lysed in 800 µl of a buffer containing 20 mM
Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1
mM benzamidine, 1 mM 1,10 phenanthroline, 10
µg/ml leupeptin, and 1 mM sodium vanadate (Sigma).
Receptors were immunoprecipitated with a polyclonal antibody to
-
and
-PDGF receptors (UBI), resolved on 7.5% polyacrylamide gels,
transferred to nitrocellulose, and exposed to autoradiographic film for
48 h. Anti-phosphotyrosine antibody 4G10 (UBI) was used for
immunoblotting.
Effects of Wortmannin
Wortmannin (Sigma) was
dissolved in MeSO to a final concentration of 10
mM, dispensed into 5-µl aliquots, and stored at -80
°C. Wortmannin aliquots were thawed and diluted 1:1000 in ice-cold
phosphate-buffered saline, and aliquots from this diluted stock were
added directly to the cells to achieve the final concentrations
indicated in each experiment. Because wortmannin is photosensitive and
unstable in aqueous solutions, it was routinely thawed, diluted, and
added to cells within 10 min. Thawed aliquots were discarded.
Initial Rates of Internalization of PDGF
Receptors
Several trafficking and sorting steps are involved in
targeting activated PDGF receptors into the lysosome. Receptors must
first clear the plasma membrane by a ligand-dependent process that
involves clustering in clathrin-coated pits, invagination, and scission
of clathrin-coated vesicles
(21) . Subsequently, receptors are
sorted to a nonrecycling, degradative pathway. The observed peripheral
distribution of receptor mutants lacking PI 3-kinase binding sites
(9) could result from a defect either in clearing the plasma
membrane or in leaving the early endosomal/recycling pathway. We
performed experiments to evaluate the ability of wild-type and mutant
receptors to undergo the initial steps of internalization.
I-labeled PDGF for 60 min at 4 °C. Unbound ligand was
washed, and cells were placed at 37 °C for 0-10 min. At
specific time points, cells were returned to 4 °C, and the ligand
remaining accessible to the extracellular space was removed by acid
washing
(20) . Uptake was only observed in cells expressing PDGF
receptors and not in cells transfected with empty pLXSN vector,
demonstrating the specificity of the assay (Fig. 1). In further
experiments internalization was measured after 10 min of incubation.
Figure 1:
Internalization of
I-PDGF. Cells were seeded in 24-well multiwell dishes and
serum-starved for 18 h. Monolayers were cooled by washing in ice-cold
DMEM and incubated for 60 min at 5 °C with 2 ng/ml
I-PDGF. Cells were then warmed to 37 °C for the times
indicated, cooled with ice-cold DMEM, and placed on ice. Cell surface
I-PDGF was removed by acid washing, and intracellular
radioactivity was determined in detergent lysates. Closedcircles, cells expressing wild-type receptors; opencircles, cells transfected with empty
vector.
Approximately 60% of the PDGF initially bound to the cell surface
(determined in cells that were not acid washed) became resistant to
acid washing after 10 min of incubation (Fig. 2). Thus, the
initial uptake of PDGF receptor in these cells was rapid and comparable
with that observed by other investigators using other cell
lines
(20) . The initial uptake of the F5 mutant was slightly but
significantly decreased compared with that of the wild-type receptor.
Add-back mutants, as well as a mutant lacking only PI 3-kinase binding
sites (F40/51), internalized PDGF like the wild-type receptor. These
results suggest that the initial steps of PDGF receptor internalization
are independent of PI 3-kinase, GAP, Syp, or phospholipase C. The
results also indicate that the large differences in the distribution of
PDGF mutant receptors in the cell, observed by
immunofluorescence
(9) , are not due to differences in the
ability of receptors to undergo the initial steps of internalization
(Fig. 2).
Figure 2:
Internalization of PDGF receptor mutants.
Cells expressing the PDGF receptor constructs indicated were seeded in
24-well multiwell dishes and serum-starved for 18 h. Monolayers were
incubated for 60 min at 5 °C with 2 ng/ml I-PDGF and
then warmed to 37 °C for 10 min. Cells were then cooled and
incubated for 5 min with phosphate-buffered saline, pH 7.4, to
determine total cell-associated ligand or with phosphate-buffered
saline, pH 3.5, to determine internalized ligand. Nonspecific binding
was determined in cells expressing the empty vector and was subtracted.
The acid-resistant (internalized) radioactivity was expressed as a
percentage of the total cell-associated radioactivity. Bars represent the mean and lines the S.E. of three
experiments performed in triplicate. Statistical comparison with the
wild-type receptor was done using paired two-tail t test
analysis. The only significant difference was between the wild-type and
the F5 receptor.
Down-regulation of PDGF Binding Sites from the Cell
Surface
Previous studies have shown that prolonged exposure of
cells to PDGF leads to a rapid disappearance of PDGF receptors from the
plasma membrane and to their subsequent degradation
(4) . This
down-regulation of PDGF receptors occurs rapidly, indicating that once
internalized, receptors must move from the early endosomal/recycling
pathway to the lysosomal pathway. The peripheral distribution of
receptor mutants lacking PI 3-kinase binding sites could be due to an
impairment in the ability of receptors to progress beyond the early
endosomal/recycling pathway. To explore this possibility, we analyzed
the extent to which PDGF receptor mutants down-regulate from the plasma
membrane. Cells expressing the indicated constructs were incubated with
unlabeled PDGF for 60 min at 37 °C and subsequently placed at 4
°C. Unlabeled PDGF was removed by a brief acid wash, and the
remaining PDGF binding sites on the cell surface were then measured
with I-PDGF. Approximately 70% of the total cell surface
binding sites for PDGF disappeared after exposure of cells expressing
wild-type receptors to 20 ng/ml of PDGF for 60 min (Fig. 3). In
contrast, only 20% of the binding sites disappeared after a similar
incubation of cells expressing the F5 mutant or a receptor lacking only
sites 740 and 751 (F40/51). Only the add-back mutant restored in PI
3-kinase binding sites (Y40/51) down-regulated to an extent similar to
that of the wild-type receptor. Thus, the ability of receptors to
efficiently down-regulate from the plasma membrane and to concentrate
in juxtanuclear structures depended on the presence of functional PI
3-kinase binding sites. Because the lack of PI 3-kinase binding sites
does not impair the initial internalization of receptors from the
plasma membrane (Fig. 2), the observed lack of efficient
down-regulation is probably due to recycling of internalized receptors
to the cell surface. Thus, lack of PI 3-kinase binding sites on the
PDGF receptor results in its failure to progress from early and/or
recycling endosomes to later steps in the endocytic pathway.
Figure 3:
Down-regulation of cell surface PDGF
receptors. Cells expressing the PDGF receptor constructs indicated were
seeded in 24-well multiwell dishes and serum-starved for 18 h. Cells
were incubated without or with 20 ng/ml PDGF-BB for 90 min at 37
°C, acid-washed with buffer at pH 3.5, and then washed twice in
DMEM to remove any remaining cell surface bound ligand and neutralize
the pH. Monolayers were then incubated for 60 min at 5 °C with 2
ng/ml I-PDGF. After washing, cell-associated
radioactivity was determined in detergent cell lysates. Nonspecific
binding was determined in cells expressing the empty vector and was
subtracted. The radioactivity associated with cells incubated with 20
ng/ml PDGF-BB was expressed as a percentage of the radioactivity
associated with cells incubated without PDGF. Bars represent
the mean and lines the S.E. of five experiments performed in
duplicate. Statistical comparison with the wild-type receptor was done
using paired two-tail t test analysis. Only the Y40/51 mutant
was not significantly different from the wild-type receptor
(NS).
Tyrosine Phosphorylation of PDGF Receptors
The
results presented above suggest that efficient down-regulation of PDGF
receptors from the cell surface requires intact PI 3-kinase binding
sites. A prediction made by this hypothesis is that receptors that fail
to down-regulate could undergo several rounds of ligand binding and
activation, and this event would be reflected by the tyrosine
phosphorylation state of the receptor. To test this prediction, we
analyzed the tyrosine phosphorylation state of PDGF receptor mutants by
immunoblotting of receptor immunoprecipitates with anti-phosphotyrosine
antibodies (Fig. 4).
Figure 4:
Tyrosine phosphorylation of PDGF receptor
mutants. HepG2 cells expressing the PDGF receptor mutants indicated
were serum-starved and then exposed to PDGF-BB (50 ng/ml) for the times
indicated. Receptors were immunoprecipitated, resolved on SDS-PAGE,
transferred to nitrocellulose, and probed with anti-phosphotyrosine
antibody. Leftpanel, an example of an immunoblot
from one experiment. Middle and rightpanels, the intensity of the phosphotyrosine signal on
the PDGF receptor bands after 5 min (middlepanel) or
120 min (rightpanel) of exposure to PDGF was
quantified by densitometric scanning. The values obtained by
densitometric scanning of four independent experiments were averaged
and plotted. The means and S.E. of these values were as follows: 76
± 13, 15 ± 6, 37 ± 16, 29 ± 9, 47 ±
6, and 39 ± 13 for WT, F5, Y40/51, Y771, Y1009 and Y1021 at 5
min and 6 ± 4, 49 ± 10, 3 ± 2, 37 ± 7, 38
± 6, 40 ± 10 at 120 min. Statistical comparison with the
wild-type receptor was done using paired two-tail t test
analysis. At 5 min, only the F5 mutant was significantly different
(p < .016) from the wild-type receptor. At 120 min, Y40/51
was not significantly different from WT, but all other mutants were
(p < .005-.05).
Tyrosine phosphorylation of wild-type and
all mutant receptors could be detected within 5 min of exposure of
cells to 50 ng/ml PDGF. The extent of tyrosine phosphorylation on the
mutant receptors was significantly lower than for the wild type after 5
min of stimulation (Fig. 4, left and middlepanels). This decrease may be due to the absence in the
mutants of three to five major autophosphorylation sites.
Phosphorylation sites remaining on the mutant receptors probably
include Tyr, a major phosphorylation site involved in
activation of the receptor kinase, as well as residues in the
juxtamembrane region of the receptor
(22, 23) . More
importantly, striking differences were observed in the kinetics of
tyrosine phosphorylation of wild-type and mutant receptors. The
phosphorylation of receptors containing PI 3-kinase binding sites
reached maximal levels after 5-10 min of incubation and then
declined. Virtually no phosphotyrosine was detected in these receptors
after 120 min of stimulation. In contrast, tyrosine phosphorylation of
the F5 mutant and of the add-back mutants containing phospholipase
C
, GAP, or Syp binding sites, increased steadily during the first
60 min of incubation. A substantial amount of phosphotyrosine (50% of
maximal value) could still be detected after 2 h of incubation with
ligand (Fig. 4, left and rightpanels). The persistence of tyrosine phosphorylation on
receptors lacking PI 3-kinase binding sites is consistent with the
possibility that these receptors undergo additional rounds of ligand
binding and activation as a consequence of recycling to the cell
surface.
Rates of Degradation of PDGF Receptors
The results
shown above indicate that a lack of PI 3-kinase binding sites on the
PDGF receptor results in its failure to progress from early and/or
recycling endosomes to later steps in the endocytic pathway leading to
the lysosome. To directly determine the rates of ligand-induced
receptor degradation, cells were labeled with
[S]methionine and then incubated for 2 h in the
absence or presence of 50 ng/ml PDGF. PDGF receptors were then
immunoprecipitated with a polyclonal antibody raised against the
receptor cytoplasmic domain. Receptors were then resolved by SDS-PAGE
and visualized by autoradiography.
Figure 5:
Degradation of PDGF receptor mutants.
HepG2 cells expressing the indicated PDGF receptor mutants were labeled
with [S]methionine and incubated for 2 h in the
absence (-) or presence (+) of PDGF-BB (50 ng/ml). Receptors
were immunoprecipitated, resolved on SDS-PAGE, transferred to
nitrocellulose, and exposed to autoradiographic film. An example of the
autoradiograph from one experiment is shown, where the major band
corresponds to the expected position of the PDGF receptor. The
increased intensity of the control band in the Y1009 lanes detected in
this experiment was not reproducible. The intensity of the PDGF
receptor bands was quantified using a Pharmacia Biotech Inc. laser
densitometer. The values obtained by densitometric scanning of three to
six independent experiments were averaged and plotted (lowerpanel). The percentage of receptor degraded after 2 h was
(mean ± S.E.) 54 ± 4, 9 ± 6, 49 ± 7, 0, 17
± 10, and 17 ± 10 for WT, F5, Y40/51, Y771, Y1009, and
Y1021, respectively. Statistical comparison was done using paired
two-tail t test analysis. Only the WT and Y40/51 mutant were
significantly degraded (p < .005 and p < .02,
respectively).
Effects of Wortmannin on PDGF Receptor
Down-regulation
The results shown above indicate a requirement
for PI 3-kinase binding sites for receptor trafficking into the
lysosomal pathway. However, these results do not prove that PI 3-kinase
is required for this trafficking process, because the same
phosphotyrosine residues that bind PI 3-kinase could conceivably also
interact with other cellular components. For example, Tyr has been shown to be a binding site for the adaptor protein
Nck
(16) . To explore the specific role of PI 3-kinase, we used a
fungal toxin, wortmannin, which has recently been shown to be a potent
inhibitor of the catalytic p110 subunit of mammalian PI
3-kinase
(18, 19) .
I-PDGF to the cell surface was measured. As
shown above, incubation of cells for 60-90 min with PDGF resulted
in the efficient down-regulation of wild-type and Y40/51 receptors but
not of the F5 mutant receptor. In contrast, when cells were treated
with wortmannin, PDGF-induced down-regulation of wild-type and Y40/51
receptors was impaired and was now indistinguishable from that of the
F5 receptor (Fig. 6). Thus, the ability of receptors that bind PI
3-kinase to efficiently down-regulate from the plasma membrane is
abolished by wortmannin treatment.
Figure 6:
Effect of wortmannin on the
down-regulation of PDGF receptors. Cells expressing wild-type
(solidbar), F5 (stripedbar), or
Y40/51 (shadedbar) mutant PDGF receptors were seeded
in 24-well multiwell dishes and serum-starved for 18 h. Cells were
incubated without or with 20 ng/ml PDGF-BB at 37 °C for the times
indicated. Where indicated, wortmannin (50 nM) was added to
cells 10 min before addition of PDGF and remained in the medium for the
rest of the experiment. Cells were cooled, acid-washed, and incubated
for 60 min at 5 °C with 2 ng/ml I-PDGF. After
washing, cell-associated radioactivity was determined in detergent cell
lysates. Nonspecific binding was determined in cells expressing the
empty vector and was subtracted. The radioactivity associated with
cells incubated with 20 ng/ml PDGF-BB was expressed as a percentage of
the radioactivity associated with cells incubated without PDGF and
plotted. This experiment was repeated three times with similar
results.
Effects of Wortmannin on PDGF Receptor
Degradation
Cells expressing wild-type PDGF receptors were
incubated with [S]methionine, chased for 45 min
with unlabeled methionine, and then incubated for 2 h in the absence or
presence of PDGF. Wortmannin, where indicated, was added to the cells
for the last 10 min of the chase period and remained in the medium for
the duration of the experiment. PDGF receptors were immunoprecipitated,
resolved by SDS-PAGE, and visualized by autoradiography (Fig. 7).
Treatment of cells with PDGF resulted in an almost complete degradation
of labeled receptor. Wortmannin fully inhibited receptor degradation in
response to PDGF but did not significantly alter the levels of labeled
receptor immunoprecipitated from cells incubated in the absence of PDGF
(Fig. 7, leftpanel). The maximal inhibitory
effect was observed between 10 and 25 nM wortmannin
(Fig. 7, rightpanel), which is similar to the
dose required to fully inhibit PI 3-kinase invitro(17, 18) . At these concentrations,
the tyrosine kinase activity of the PDGF receptor was unaffected (not
shown). Thus, the down-regulation and degradation of PDGF receptors in
human HepG2 cells is fully blocked in response to concentrations of
wortmannin that inhibit PI 3-kinase. These results support the
hypothesis that the requirement for PI 3-kinase binding sites for PDGF
receptor trafficking into the lysosomal pathway is due to a requirement
for PI 3-kinase activity.
Figure 7:
Effect of wortmannin on the degradation of
wild-type PDGF receptors. HepG2 cells expressing the wild-type PDGF
receptor were labeled with [S]methionine and
incubated for 2 h in the absence (-) or presence (+) of
PDGF-BB (50 ng/ml). Wortmannin was added at the concentrations
indicated 10 min before exposure to PDGF. A representative
autoradiograph of the receptor immunoprecipitates is shown in the
leftpanel. The expected position of the PDGF
receptor is indicated with an arrow. The intensity of the PDGF
receptor band was quantified with a Pharmacia laser densitometer, and
the results were plotted (rightpanel).
-factor but is critically
required for its subsequent delivery into the vacuole and its
degradation
(27) . These results are consistent with a direct
role for PI 3-kinase in sorting in the endocytic pathway both in yeast
and mammalian cells.
, and PI-3,4,5-P
,
may have unique targets and thus coordinately but independently
regulate multiple cellular responses. In contrast to mammalian PI
3-kinase, yeast vps34 only phosphorylates PI to produce PI-3-P,
suggesting that this lipid may be the principal product involved in
protein sorting events. In summary, genetic and pharmacological
evidence presented here suggests a critical requirement for PI 3-kinase
activity in postendocytic trafficking and down-regulation of
-PDGF
receptors. Important questions remain relating to the biochemical basis
for this requirement.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.