From the Departments of Biological Regulation and
§ Molecular Cell Biology, The Weizmann Institute of Science,
Rehovot 76100, Israel
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ABSTRACT |
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The ErbB signaling module consists of four
receptor tyrosine kinases and several dozen ligands that activate
specific homo- and heterodimeric complexes of ErbB proteins.
Combinatorial ligand/receptor/effector interactions allow large
potential for signal diversification. Here we addressed the possibility
that turn-off mechanisms enhance the diversification potential.
Concentrating on ErbB-1 and two of its ligands, epidermal growth factor
(EGF) and transforming growth factor (TGF-
), and the Neu
differentiation factor (NDF/neuregulin) and one of its receptors,
ErbB-3, we show that ligand binding variably accelerates endocytosis of
the respective ligand-receptor complex. However, unlike the
EGF-activated ErbB-1, which is destined primarily to degradation in
lysosomes, NDF and TGF-
direct their receptors to recycling,
probably because these ligands dissociate from their receptors earlier
along the endocytic pathway. In the case of NDF, structural, as well as
biochemical, analyses imply that ligand degradation occurs at a
relatively late endosomal stage. Attenuation of receptor
down-regulation by this mechanism apparently confers to both NDF and
TGF-
more potent and prolonged signaling activity. In conclusion,
alternative endocytic trafficking of ligand-ErbB complexes may tune and
diversify signal transduction by EGF family ligands.
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INTRODUCTION |
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The ErbB/HER family of receptor tyrosine kinases is representative of several other groups of highly homologous receptors sharing growth factors as their ligands (1). Whereas only one ErbB homolog exists in invertebrates, such as flies and worms, four receptors are present in mammals, and they bind to multiple growth factor molecules through an epidermal growth factor (EGF)1 motif. The variety of receptors and ligands is thought to contribute to the diversification potential of the ErbB signaling network (reviewed in Ref. 2). Three levels of signal diversification may be proposed. First, each ligand molecule is apparently bivalent; the high affinity binding site binds to a "primary receptor," whereas a low affinity site, whose specificity is broad, allows selective recruitment of ErbB molecules into dimeric receptor structures, leading to effective signaling (3). Second, at the receptor level, 10 distinct homo- and heterodimeric complexes may be formed in a ligand-specific order of preference. Yet, the most divergent level of the signaling mechanism appears to be postreceptor; through their various autophosphorylation sites, dimeric receptor complexes engage specific sets of cytoplasmic signaling proteins sharing a phosphotyrosine binding motif, usually a Src homology 2 domain (4).
Despite the remarkably large repertoire of signaling proteins that
become physically associated with specific ErbB dimers, only a few have
been shown to be strictly specific. Examples include the
phosphatidylinositol 3'-kinase, an enzyme that selectively binds to
ErbB-3-containing dimers (5), and phospholipase C, a specific target
of ErbB-1 (6) and ErbB-2 (7). Moreover, signaling pathways stimulated
by all ErbB combinations feed into the mitogen-activated protein kinase
(MAP kinase) pathway (8), resulting in a surprisingly uniform pattern
of signaling, which is shared by the invertebrate versions of ErbB
proteins (9). Nevertheless, unlike the shared rapid coupling to MAP
kinases, the kinetics of MAP kinase inactivation, a critical parameter in determining signal outcome (10), differ according to the identity of
the stimulating receptor. Especially important is the engagement of
ErbB-2, a ligandless protein that delays inactivation of ErbB signals
(11), and ErbB-3, a kinase-defective receptor whose activation depends
on the presence of a co-receptor (8, 12). Thus, the inactivation phase
of ErbB signaling may be critical for determination of the duration and
potency of cell activation by EGF family growth factors.
Whereas the major events involved in ErbB activation are fairly well
understood, the inactivation phase is still poorly characterized. Several lines of evidence, however, suggest that turn-off mechanisms may be as important for signal diversification as is the stimulatory process. An obvious candidate is the interaction of ligand-occupied receptors with tyrosine-specific phosphatases (13). Cbl is another example; this cytoplasmic adaptor protein, whose ortholog in
Caenorhabditis elegans negatively regulates ErbB signaling
(14), associates with ErbB-1 but not with ErbB-3 or with ErbB-4 (15).
The third mechanism of receptor inactivation involves ligand-induced
down-regulation of the number of surface receptors. This fairly complex
regulatory process is mediated by rapid endocytosis of ligand-receptor
complexes, and their variable delivery into cytoplasmic compartments
(for review see Ref. 16). Unlike receptors for nutrients such as transferrin and lipoproteins, efficient internalization of growth factor receptors through coated pits requires ligand binding. In
addition, a more significant fraction of the endocytosed signaling receptors escapes recycling and is sorted to degradation in lysosomes. Apparently, a fine balance between receptor recycling and lysosomal targeting exists in cells, yet its molecular details are still poorly
understood. For example, specific domains of ErbB-1 are necessary for
lysosomal targeting (17), and certain cytoplasmic proteins, such as
phosphatidylinositol 3'-kinase, are probably involved in the selective
sorting of internalized receptors (18). Another critical parameter is
the pH sensitivity of ligand/receptor interactions; ligands, such as
EGF, whose interaction is relatively resistant to mildly acidic pH,
target ErbB-1 to lysosomes, whereas transforming growth factor (TGF-
) and other ligands that readily dissociate due to the mildly
acidic endosomal pH cause receptor recycling (19). Despite the open
questions, it seems clear that endocytosis and lysosomal targeting
critically attenuate signaling by growth factors (20, 21).
Recent reports suggest that alternative intracellular routing of ErbB
proteins and their ligands may contribute to diversification of signal
transduction (8, 22). In contrast to the rapid internalization of
ErbB-1 and its ligand, EGF, retarded endocytosis of the three other
members of the ErbB family has been observed. In this paper, we have
investigated the mechanism underlying differential receptor trafficking
by ErbB ligands. We have found that an ErbB-3-specific ligand, the Neu
differentiation factor (NDF/neuregulin), undergoes slow endocytosis
that is followed by receptor recycling to the plasma membrane. By
contrast, most of the EGF-stimulated ErbB-1 molecules are destined to
lysosomal degradation. Due to the consequent clearance of ErbB-1
molecules, but not ErbB-3 molecules, from the cell surface, the
mitogenic signal evoked by EGF is less potent than the NDF signal. This
phenomenon, however, depends also on the nature of the ligand, since
another ErbB-1 ligand, TGF-, undergoes rapid endocytosis but
nevertheless induces receptor recycling with only limited receptor
down-regulation. Consistently, the mitogenic activity of this ligand is
superior to that of EGF, reinforcing a role for ErbB routing in tuning
of mitogenic signals.
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EXPERIMENTAL PROCEDURES |
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Materials, Buffers, and Antibodies--
A recombinant form of
NDF-1177-246 was prepared by Amgen (Thousand Oaks, CA).
Human recombinant EGF and TGF-
were purchased from Sigma.
Radioactive materials were purchased from Amersham Pharmacia Biotech.
IODO-GEN was from Pierce. A monoclonal antibody to ErbB-3 was generated
in our laboratory (23). The ErbB-1-specific monoclonal antibody 528 was
a gift from John Mendelsohn (M. D. Anderson Cancer Center). The
monoclonal antibody to the active form of MAP kinase (doubly
phosphorylated on both tyrosine and threonine residues of the TEY motif
(24)) was a gift from Rony Seger (Weizmann Institute, Rehovot, Israel).
Binding buffer contained Dulbecco's modified Eagle's medium with
0.5% bovine serum albumin and 20 mM HEPES. HNTG buffer
contained 20 mM HEPES (pH 7.5), 150 mM NaCl,
0.1% Triton X-100, and 10% glycerol. Solubilization buffer contained
50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10%
glycerol, 1% Nonidet P-40, 1.5 mM EGTA, 2 mM
sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride,
aprotinin (0.15 trypsin inhibitor unit/ml), and 10 µg/ml leupeptin.
Biotin-X-NHS was from Calbiochem. Colloidal gold sols (10-nm diameter)
were from British Biocell International. All other chemicals were
purchased from Sigma, unless otherwise indicated.
Cell Lines-- The Chinese hamster ovary (CHO) cell lines expressing the various ErbB proteins or a deletion mutant of ErbB-3 were described previously (25). The ErbB-3 QQGFF insertion mutant was generated by oligonucleotide-directed mutagenesis using T7-DNA polymerase (26). The cDNA of erbB-3 was subcloned into the pCDNA3 expression vector containing an F1 origin of replication, and the mutation was introduced by using the primer 5'-GGAGCAGGAGTTAGAAAAAGCCCTGTTGCGTTCTCTGGG-3' and the purified single-stranded DNA as a template. Cells were cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with antibiotics and 10% heat-inactivated bovine serum. For immunoprecipitation and immunoblotting experiments, cells were grown to 90% confluence. Sublines derived from the 32D murine hematopoietic progenitor cell line and expressing various ErbB proteins have been previously described (8). Cells were grown in RPMI 1640 medium supplemented with antibiotics, 10% heat-inactivated fetal bovine serum, and 0.1% medium that was conditioned by an interleukin-3-producing cell line (27).
Radiolabeling of Ligands and Ligand Binding Analyses--
Human
recombinant EGF, TGF-, and human recombinant
NDF-
1177-246 were labeled with IODO-GEN (Pierce) as
described (3). For binding assays, monolayers of the indicated cell
lines in 24-well dishes were washed once with binding buffer and then
incubated for 2 h at 4 °C with 10 ng/ml
125I-labeled NDF-
1177-246,
125I-EGF, or TGF-
. The cells were washed three times
with ice-cold binding buffer. Labeled cells were lysed for 15 min at
37 °C in 0.5 ml of 0.1 N NaOH solution containing 0.1%
SDS, and the radioactivity was determined. Nonspecific binding was
calculated by subtracting the binding of radiolabeled ligands to CHO
cells or by performing the binding assays in the presence of a 100-fold
excess of unlabeled ligand.
Ligand Internalization Assay-- Cells cultured in 24-well plates were washed with binding buffer and then incubated for 2 h at 4 °C in the presence of radiolabeled NDF or EGF (each at 10 ng/ml). To allow ligand internalization, the cells were transferred to 37 °C and incubated for various periods of time. The cells were then put on ice and washed twice with binding buffer, and cellular distribution of the radiolabeled ligand was determined by using a 7-min-long incubation in 0.5 ml solution of 150 mM acetic acid (pH 2.7), containing 150 mM NaCl. The released ligand was considered as cell surface-associated ligand (28). The remaining radioactivity was solubilized in 100 mM NaOH solution containing 0.1% SDS that was considered as internalized ligand.
Receptor Down-regulation Assay-- Ligand-induced receptor down-regulation was measured as follows. Cells grown in 24-well plates were incubated at 37 °C for up to 2.5 h without or with 250 ng/ml ligand in binding buffer and then rinsed with cold binding buffer. Surface-bound growth factor was removed by using a low pH acetic acid wash. The number of ligand binding sites on the cell surface was then determined by incubating cells at 4 °C with the corresponding radiolabeled ligand for at least 1 h.
Electron Microscopy--
Coupling of EGF and NDF to gold
colloids (10-nm diameter) was performed by using a standard protocol
provided by the manufacturer. Cellular uptake of colloidal gold-labeled
ligands was performed as follows. Cells grown in 24-well plates were
rinsed twice with binding buffer at 4 °C and then incubated in the
same buffer containing 1:10 dilution of either NDF- or EGF-colloidal
gold for 45 min at 4 °C. The cells were then warmed to 37 °C and
incubated for various time intervals in the continuous presence of the
ligand. At the end of incubation at 37 °C, cells were rinsed twice
with binding buffer and fixed for 24 h with Karnovsky's fixative
(3% formaldehyde, 2% glutaraldehyde, and 5 mM
CaCl2 in 100 mM cacodylate buffer, pH 7.4) at
room temperature and 10 h at 4 °C. Lighter fixation was then
carried out with a mixture of 3% formaldehyde and 0.1% glutaraldehyde
in the same buffer. Tissues were impregnated in 100 mM
cacodylate buffer, with 2.3 M sucrose, pH 7.4. The samples were quickly frozen in liquid nitrogen, and ultrathin sections (500-1000 Å) were cut at 115 °C using a Reichert-Jung FS-4D
ultracryomicrotome. The sections were recovered from the knife in a 2.3 M sucrose droplet according to the method of Tokuyasu and
Singer (29) and transferred to Formvar-coated 300-mesh grids. The grids
were floated on water in a Petri dish at 4 °C. The duration of the water rinse was between 1 and 16 h, without any appreciable
difference in the resulting ultrastructure. The sections were then
processed for methyl-cellulose mounting according to a modification of
the above mentioned protocol.
Cell Proliferation Assay--
Cells were washed free of
interleukin-3, resuspended in RPMI 1640 medium at 5 × 105 cells/ml, and treated without or with EGF, TGF-, or
NDF at various concentrations for dose response experiments. Cell
survival was determined by using the
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay, which determines mitochondrial activity in living cells (30).
MTT (0.1 mg/ml) was incubated with the analyzed cells for 2 h at
37 °C. Living cells can transform the tetrazolium ring into dark
blue formazan crystals that can be quantified by reading the optical
density at 540-630 nm after lysis of the cells with acidic isopropyl
alcohol.
Cell Surface Biotinylation-- Monolayers of CHO cells grown in 10-cm dishes were washed three times with ice-cold phosphate-buffered saline and then incubated with 0.5 mg/ml of a water-soluble Biotin-X-NHS (Calbiochem) dissolved in borate buffer (10 mM boric acid, 150 mM NaCl, pH 8.0) for 45 min at 4 °C. Coupling of biotin was blocked by extensive washes with a solution of 15 mM glycine in phosphate-buffered saline. Cells were then treated with a growth factor (250 ng/ml) for different time intervals at 37 °C. To evaluate the amounts of cell surface receptors, the cells were subjected to immunoprecipitation and gel electrophoresis. Visualization of the biotinylated proteins was performed by probing the nitrocellulose membranes with horseradish peroxidase-conjugated streptavidin (Amersham Pharmacia Biotech) and developed with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).
Lysate Preparation and Immunoprecipitation-- After treatment with growth factors, cells were scrapped, pelleted by centrifugation, and solubilized in solubilization buffer. Lysates were cleared by centrifugation (10,000 × g, 10 min). For direct electrophoretic analysis, boiling gel sample buffer was added to cell lysates. For other experiments, lysates were first subjected to immunoprecipitation with anti-receptor monoclonal antibodies that were precoupled to rabbit-anti-mouse immunoglobulin G and then to protein A-Sepharose. The proteins in the lysate supernatant were immunoprecipitated with aliquots of the protein A-Sepharose-antibody complex for 2 h at 4 °C. The immunoprecipitates were washed three times with HNTG, resolved by SDS-polyacrylamide gel electrophoresis through 7.5% gels, and electrophoretically transferred to nitrocellulose membranes. Membranes were blocked for 2 h in TBST buffer (0.02 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20) containing 0.1% milk, blotted with 1 µg/ml primary antibodies for 2 h, followed by 0.5 µg/ml secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected with the ECL (enhanced chemiluminescence) reagent (Amersham Pharmacia Biotech).
Analysis of Growth Factor Degradation-- Conditioned media containing radiolabeled ligands were assayed by the addition of cold trichloroacetic acid (10% final concentration). Samples were precipitated after 1 h at 2 °C by centrifugation for 15 min at 4 °C. Radioactivity present in the supernatants (trichloroacetic acid-soluble fraction) and pellets (trichloroacetic acid-insoluble fraction) was determined.
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RESULTS |
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NDF and EGF Receptors Are Differentially Endocytosed and
Metabolized following Ligand Binding--
It has been previously
reported that the rates of ligand-induced internalization of NDF
receptors are significantly slower than the rate of EGF-driven
endocytosis of ErbB-1 (8, 22). We used a series of CHO cells (25),
which share landmarks with many ErbB-expressing epithelial and
fibroblastic cells, to address the mechanism underlying differential
endocytosis of ErbB proteins. Comparative analysis revealed that CHO
cells ectopically expressing ErbB-1 (denoted CB1 cells) internalized
radiolabeled EGF at a higher rate than the kinetics of endocytosis of
surface-bound NDF into cells expressing ErbB-3 (CB3 cells) or ErbB-4
(CB4 cells, Fig. 1A). Two
lines of evidence indicated that this difference is receptor-specific
rather than ligand-specific. First, internalization of EGF by cells
expressing a chimeric receptor, denoted NEC, in which the extracellular
and intracellular domains are derived from ErbB-1 and ErbB-2,
respectively (31), was relatively slow, in accordance with a previous
report (32). Second, the rate of internalization of another ErbB-1
ligand, namely TGF-, was comparable with that of EGF (Fig.
1A). These results are consistent with previous reports (8,
22, 33), and they imply that the differences in cellular uptake of NDF
and EGF are independent of cell type or ligand identity.
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NDF and EGF Undergo Differential Intracellular Metabolism, Probably
Due to Distinct pH Sensitivity of Ligand/Receptor
Interactions--
Mutations in several types of receptors, including
the low density lipoprotein receptor (38) and the insulin receptor
(39), accelerate ligand degradation by stabilizing the ligand-receptor complex at the low pH (pH ~6.2) of endosomes (40).
Likewise, the pH sensitivity of ErbB-1/ligand interactions apparently
affects the balance between recycling and degradation of EGF family
ligands (19). To determine whether differential dissociation of NDF, EGF, and TGF- can account for alternative routing of their
receptors, we tested the stability of the corresponding ligand-receptor
complexes at various pH conditions. As is evident from Fig.
4, NDF, like TGF-
, displayed higher pH
sensitivity than EGF at the relevant range of pH values, supporting a
model attributing recycling of ErbB-3 to NDF-ErbB-3 dissociation in
early endosomes.
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Subcellular Distribution of Internalized NDF-- Endocytosed molecules are first delivered to early endosomes, which are tubular and vesicular membrane structures often connected to networks and located at the cell periphery. EGF and its receptor can be detected in this compartment within 2-5 min of internalization onset (44). After 10-15 min, EGF-occupied ErbB-1 molecules begin to accumulate in large tubular-vesicular endosomes, located mainly around the nucleus. EGF and ErbB-1 become detectable within lysosomes after 30-60 min of internalization, but they are retained in these compartments for several hours. To follow the subcellular distribution of internalized NDF, in comparison with EGF, we examined the distribution of colloidal gold-labeled NDF and EGF in CB3 and in CB1 cells, respectively, by using electron microscopy. In cells that were incubated with NDF-gold particles at 4 °C, NDF was distributed almost exclusively on the plasma membrane, and it displayed either homogeneous distribution or local clusters (Fig. 6A). Within 5 min of transfer to 37 °C, most of the membrane-localized NDF disappeared, but the labeled ligand could be detected in relatively small vesicles, corresponding to early endosomes, that were located throughout the cytoplasm (Fig. 6B). Upon longer incubation (50 min) of cells at the elevated temperature, NDF became associated with larger elongated vesicular structures with a perinuclear location. The irregular morphology of these vesicles and the vesicular content of some structures (Fig. 6C, inset) suggest they are multivesicular bodies. By contrast, at this time point colloidal gold-labeled EGF was observed mainly in relatively large and uniform vesicles with electron dense content, consistent with their identification as lysosomes (Fig. 6D). Surprisingly, at this time point, gold-labeled NDF reappeared at the plasma membrane (Fig. 6C). Similar reappearance was not observed in the case of gold-labeled EGF, and quantitative analysis of the number of membrane-associated grains in comparison with earlier time points indicated it was statistically significant (p < 0.023). The reappearance of NDF at the plasma membrane probably reflects recycling of ligand-receptor complexes, or the formation of new complexes between the extracellular NDF-gold and newly maturing or recycled receptors. This conclusion is consistent with biochemical indications of ErbB-3 recycling and rapid recovery (Figs. 2 and 3). Taken together, the results presented in Fig. 6 provide structural support to the proposition based upon biochemical assays (Figs. 1 and 5) that NDF undergoes endocytosis and its degradation occurs relatively late in the endocytic pathway, namely in nonlysosomal multivesicular bodies (see also Fig. 5).
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Mutational Analysis of ErbB-3 Endocytosis--
Several alternative
explanations to the slow endocytosis of ErbB-3 may be proposed. First,
endocytosis may be impaired due to the defective kinase of ErbB-3 (45).
However, the fact that ErbB-4, whose kinase is active, shares slow
kinetics with ErbB-3 (Fig. 1) does not support this explanation.
Alternatively, because ErbB-3 does not interact with adaptor protein 2 (AP-2) of the clathrin-coated pit, but ErbB-1 efficiently associates
with AP-2 (22), endocytosis of ErbB-1 is more rapid. According to a
third model, an endocytosis-directing sequence motif, namely the QQGFF signal (46), which is found on ErbB-1 but not on ErbB-3, accounts for
rapid endocytosis of ErbB-1. To experimentally examine this model, we
constructed and transiently expressed two mutants of ErbB-3: a deletion
mutant lacking the whole intracellular domain (denoted TAG-3M; Ref. 25)
and a full-length ErbB-3 containing an ectopic QQGFF sequence at its C
terminus (QQGFF-ErbB-3). Fig. 7 depicts
the results of an NDF internalization assay performed with cells
expressing the two mutants, or wild type ErbB-3. Evidently, introduction of the endocytic signal of ErbB-1 into ErbB-3 did not
alter the rate of endocytosis of this NDF receptor, suggesting that the
reason for the slow endocytosis may not be the absence of the QQGFF
motif in ErbB-3. Likewise, deletion of the entire cytoplasmic portion
of ErbB-3 only slightly affected the rate of NDF endocytosis but
enhanced the overall extent of ligand internalization. Thus, it seems
that a cytoplasmic sequence(s) of ErbB-3 can retard internalization,
but its inhibitory action cannot be released by a sequence code
containing a -turn configuration necessary for rapid
endocytosis.
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The Relative Biological Potencies of ErbB Ligands Correlate with
Their Endocytic Fates--
The emerging differential trafficking of
ligand-receptor complexes, and especially the relatively limited
receptor down-regulation observed after cell stimulation with either
NDF or TGF-, prompted us to determine the implications to biological
activities. Because CHO cells exhibit some characteristics of
transformed cells, including tumor formation in athymic mice, we used
as a cellular system an interleukin 3-dependent 32D myeloid
cell line that allows sensitive detection of growth signals.
Importantly, 32D cells share with CHO cells differential endocytosis of
NDF and EGF receptors, including features like ligand and receptor
degradation, receptor down-regulation, and recovery of binding sites
after exposure to the respective ligand (Ref. 8 and data not shown). A
derivative 32D line, denoted D13, that co-expresses ErbB-1 and ErbB-3
was stimulated with NDF, TGF-
, or EGF, and cell proliferation tested
by using the MTT assay (30). The resulting dose-response curves
indicated that NDF is the most potent of the three ligands, whereas
EGF, whose effect on receptor down-regulation was most prominent (Fig. 2), is the least active mitogen in this model cell system (Fig. 8A). To correlate these
results with possible kinetic differences in cell activation, we
examined the respective patterns of receptor phosphorylation by using
antibodies to phosphotyrosine (Fig. 8B). In experiments that
are not presented, we found that mostly ErbB-3 was phosphorylated in
response to NDF, and both EGF and TGF-
induced exclusive
phosphorylation of ErbB-1. Likewise, MAP kinase activation was tested
by utilizing an antibody specific to the active form of Erk (24) (Fig.
8C). Evidently, all three ligands rapidly activated receptor
phosphorylation, but unlike the relatively weak activation observed
with EGF, potent and persistent stimulation was displayed by both NDF
and TGF-
. Surprisingly, MAP kinase activation by NDF occurred after
a lag period of 1-2 min, but activation, especially of the p44 isoform
of Erk, was more prolonged relative to EGF. TGF-
evoked an
intermediate pattern of MAP kinase activation, consistent with its
moderately high effect on cell proliferation (Fig. 8A).
Conceivably, the combination of slow endocytosis with limited
down-regulation and extensive recycling of ErbB-3 prolongs cell
activation through the NDF·ErbB-3 complex, thereby allowing more
potent mitogenic signals.
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DISCUSSION |
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The comparative biochemical and structural analyses of
ligand-mediated endocytosis of ErbB-3 and ErbB-1 we have presented in
this report indicate differences between the two pathways at all steps
of ligand/receptor processing, including the initial internalization
step at the cell surface (Figs. 1 and 2), dissociation of the complexes
in an endocytic compartment (Fig. 4), receptor recycling to the plasma
membrane (Figs. 2 and 3), and degradation of both the ligand (Fig. 5)
and the receptor (Fig. 1). Whereas NDF and EGF are sorted into distinct
pathways, TGF- displays a composite pathway; initially it follows an
EGF-characteristic route, but later it seems to enter the NDF pathway.
In some respects, the routing of ErbB-3 is analogous to the one taken
by a kinase-defective ErbB-1 (41, 47, 48), implying that the impaired
kinase activity of ErbB-3 is responsible for the alternative routing of
this receptor. However, closer inspection of the data suggests
involvement of additional parameters, which are discussed below. Fig.
9 schematically depicts the inferred
distinct intracellular routes of NDF and EGF, and the following
discussion highlights their major attributes.
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Formation of Early Endosomes--
Initially, NDF binds to surface
ErbB-3 molecules (Fig. 6), whose distribution is largely similar to the
pattern of surface ErbB-1 that was observed with ferritin-labeled EGF
molecules (49). Within minutes at 37 °C, NDF is transferred to small
and relatively clear vesicles containing 1-20 NDF-receptor complexes
(Fig. 6). Despite morphological similarities to EGF internalization,
removal of NDF from the cell surface is much slower than the rate of
EGF or TGF- internalization (Fig. 1). Not only is ErbB-3 a slowly internalizing receptor, but also ErbB-2 and ErbB-4 differ from ErbB-1
in their rates of endocytosis (Fig. 1 and Ref. 22). Thus, entry appears
to be receptor-specific rather than ligand-specific, and its rate may
be independent on kinase activity. Analyses of a kinase-defective
mutant of ErbB-1 indicated that its rate of internalization is smaller
than that of the wild type receptor (41, 48, 50, 51). Therefore, a
causative role of the intrinsic kinase function in fast endocytosis
cannot be ruled out. Conceivably, a receptor-specific sorting machinery
exists at an early endocytic stage. It is unknown whether or not the
same mechanism also controls the basal receptor turnover rate, which is
fast in the case of ErbB-3 and slow with ErbB-1 (Fig. 1). Mutational
analyses of ErbB-1 have identified sequence motifs that may be
responsible for differential sorting. Thus, two motifs, QQGFF and
FYRAL, found in the C terminus of ErbB-1, could effectively replace the
endocytic tyrosine-containing code of the transferrin receptor (46).
Tyrosine 974 of the latter motif was shown to mediate AP-2 interactions
(52). Whereas several motifs in ErbB-3 meet the FYRAL predicted
structure, no analog of a QQGFF motif is found in the carboxyl-terminal
tail of this receptor. Grafting this motif, however, into ErbB-3 did
not recover rapid endocytosis (Fig. 7). Possibly, an active kinase is
essential for the function of this motif in ErbB-3, as it is in the
case of ErbB-1 (46), or additional sequences are involved.
Receptor Sorting in Endosomes--
Approximately 15 min after
binding to ErbB-3, NDF can be detected in large tubular-vesicular
endosomes located close to the nucleus (Fig. 6). EGF follows an
apparently similar pathway to reach an extensive network of tubular
cisternae (49, 53). Immunocytochemical evidence suggests that the
vacuolar structures with tubular arms are an important site of
ligand/receptor dissociation (54). However, recycling of internalized
receptors also occurs before they reach the multivesicular bodies;
recycling from early endosomes is even faster than recycling from
multivesicular bodies (reviewed in Ref. 16). Our structural data
indicate that NDF reaches multivesicular bodies, but we were unable to
determine whether at this step the ligand is still complexed to ErbB-3. Nevertheless, because the endosomal pH gradually decreases along the
endocytic pathway and NDF dissociates at a relatively mild acidic pH
(Fig. 4), it is conceivable that recycling of ErbB-3 occurs primarily
prior to the late endosomal stage of multivesicular bodies. In fact,
several biochemical lines of evidence indicate that recycling of ErbB-3
is much more extensive than that of an EGF-driven ErbB-1. Thus,
exposure to high NDF concentrations resulted in a relatively moderate
disappearance of ErbB-3. Moreover, inhibition of recycling with
monensin enhanced the extent of ErbB-3 down-regulation, but it did not
affect ErbB-1 (Fig. 2). Likewise, recovery of ErbB-3 after a
down-regulation pulse is rapid and exceeds the original receptor level,
indicative of accelerated delivery of receptors from internal pools
(Fig. 3). The reappearance of a binding-competent ErbB-3 at the cell
surface was detectable also by using electron microscopy (Fig.
6C). The most likely explanation to the extensive recycling
of ErbB-3 is the relatively acid-sensitive NDF/ErbB-3 interaction.
Presumably, an intact EGF·ErbB-1 complex mediates phosphorylation of
an endosomal substrate that actively directs ErbB-1 to a lysosomal
destination, but inactivation of the TGF-·ErbB-1 complex due to
ligand dissociation prevents phosphorylation of the critical substrate.
In the case of the NDF·ErbB-3 complex, phosphorylation may not occur
either because the kinase is inactive or because NDF dissociates from
ErbB-3-containing heterodimers already at an early endocytic step. This
model is compatible with the correlation reported between the pH
sensitivity of several ErbB-1-specific ligands and their intracellular
trafficking (19). In conclusion, recycling of ErbB-3 after NDF binding
is analogous to the routing of a kinase-defective mutant of ErbB-1,
whose down-regulation is defective and its recycling is
enhanced (41).
Ligand Degradation--
Despite remarkable differences in their
cellular routing, NDF, EGF, and TGF- undergo efficient and
comparable proteolytic degradation (Fig. 5). This observation is in
agreement with two recent reports that observed intracellular
degradation of NDF (33) and TGF-
(43). However, unlike the well
characterized degradation of EGF in mature lysosomes (Fig. 6) (for a
review see Ref. 16), proteolysis of NDF and TGF-
apparently takes place in a compartment distinct from the site of EGF degradation. Our
results indicate that the unknown site of NDF degradation is partly
sensitive to chloroquine, a drug affecting vesicle processing at a
prelysosomal site (42), but resistant to the lysosome-specific drug
leupeptin (Fig. 5). Similar characteristics are shared by the
degradative pathway of TGF-
(see Fig. 5 and Ref. 43). Because we
observed NDF in multivesicular bodies, but not in lysosomes, it is
conceivable that NDF degradation occurs in a nonlysosomal endocytic
compartment.
Relationships between Intracellular Routing and Signaling
Potency--
Several lines of evidence support the notion that
receptor endocytosis attenuates mitogenic signaling by down-regulating
the receptor and by depleting the ligand from the extracellular space. An endocytosis-defective receptor for EGF delivers more potent mitogenic and oncogenic signals than the wild-type ErbB-1 (20), and an
EGF mutant that cannot down-regulate its receptor due to low binding
affinity and sensitivity to acidic pH elicits stronger mitogenic
signals than EGF and TGF- (21). In many cellular systems, such as
keratinocytes (55) and endothelial cells (56), TGF-
is a more potent
ligand than EGF. This has been attributed to the impaired ability of
TGF-
to induce receptor down-regulation (34). Employing an
interleukin-dependent myeloid cell line, we confirmed
superiority of TGF-
, relative to EGF, but found that NDF is an even
better mitogen for the same type of cells (Fig. 8). Although the
greater potency of NDF may be explained by a better selection of
signaling proteins by the multiple docking sites of ErbB-3 (57), the
analogy with TGF-
favors the possibility that the relatively slow
internalization of ErbB-3 and its extensive recycling are involved in
signal amplification. Presumably, retarded depletion of NDF from the
medium, along with the continuous presence of ErbB-3 on the cell
surface, allows prolongation of signal transduction through the MAP
kinase pathway (Fig. 8C). Many studies performed with well
defined systems suggest that prolonged activation of the MAP kinase
pathway can lead to oncogenic transformation or to cellular
differentiation, depending on the cellular context (10). In the case of
NDF, the relatively persistent signaling by this ligand may explain its
ability to act as either a differentiation factor (58) or a very potent
mitogen (8, 12).
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ACKNOWLEDGEMENTS |
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We thank Barry Ratzkin (Amgen, Thousand Oaks, CA) for the recombinant NDF preparation, Ronit Pinkas-Kramarski and Eldad Tzahar for establishment of cell lines, and Rony Seger and John Mendelsohn for monoclonal antibodies.
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FOOTNOTES |
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* This work was supported by the Susan G. Komen Breast Cancer Foundation; NCI, National Institutes of Health Grant CA72981; and the Israel Science Foundation.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: Dept. of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-9343974; Fax: 972-8-9344125; E-mail: liyarden{at}weizmann.weizmann.ac.il.
1
The abbreviations used are: EGF, epidermal
growth factor; CHO, Chinese hamster ovary, MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NDF, Neu
differentiation factor; TGF-, transforming growth factor
; MAP,
mitogen-activated protein.
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REFERENCES |
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