(Received for publication, June 26, 1996, and in revised form, October 28, 1996)
From the Departments of The RET ( The RET gene encodes a receptor tyrosine kinase homolog
(1, 2). RET was first identified in DNA transfection studies in which the gene had been Inactivating mutations in RET result in Hirschsprung disease
(9-11). Hirschsprung disease is characterized by megacolon resulting from a failure to innervate the gut. RET knockout mice also
exhibit megacolon, confirming the involvement of RET
in this symptom, but their most remarkable phenotype is renal agenesis
(12). The association of mutations in RET in neoplasia
as well as Hirschsprung disease suggests that this tyrosine kinase is
important in cell proliferation and development.
Because the ligand for Ret has only recently been identified (13-17),
chimeric receptors containing the extracellular domain of the
EGF1 receptor and the intracellular domain
of Ret have been used to characterize the ligand-activated form of the
Ret tyrosine kinase. Santoro et al. (18) expressed an
EGFR/RET chimera in NIH 3T3 cells and reported that EGF stimulated the
tyrosine kinase activity of EGFR/RET and enhanced the exchange of GDP
for GTP on Ras in these cells. However, the chimeric receptor failed to
stimulate MAP kinase activity or phosphatidylinositol 3-kinase
activity. van Weering et al. (19) expressed a similar
EGFR/RET chimera in SK-N-MC cells, a neuroectodermal cell line. In this
system, EGF stimulated the tyrosine kinase activity of the EGFR/RET
chimera and also led to the sustained activation of MAP kinase. The
authors suggested that the differences observed between their cell
lines and those of Santoro et al. (18) indicated a cell
type-specific ability of Ret to stimulate MAP kinase activation.
Despite the fact that EGF failed to stimulate MAP kinase in 3T3 cells
expressing the EGFR/RET chimera, this growth factor did induce both
proliferation and transformation of these cells (18), suggesting that
activation of MAP kinase may not be required for Ret to promote
proliferation of 3T3 cells.
In this report, we functionally characterize a chimeric EGFR/RET
receptor expressed in NIH 3T3 cells with respect to its ligand binding
and internalization properties. In addition, regulation of the EGFR/RET
chimera via receptor down-regulation and protein kinase
C-dependent mechanisms was investigated. The findings
suggest that many of the common mechanisms used to terminate signaling via growth factor receptors do not function in the Ret tyrosine kinase
system. The absence of such termination events suggests that prolonged
activation of Ret and/or a maintenance of sensitivity to Ret ligand may
be physiologically important for cells that normally express this
growth factor receptor.
Materials
NIH 3T3 cells expressing wild type EGF receptors were as
described previously (20). Antibodies to Ret and Ras-GAP were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphotyrosine antibodies (PY-20) were from Transduction Laboratories.
[35S]Methionine was from Amersham. Polyclonal antibodies
against the EGF receptor (DB1) and 125I-EGF were prepared
as described previously (21). Western blots were developed using
enhanced chemiluminescence reagents from Amersham. All other reagents
were from Sigma Chemical Co.
Methods
The extracellular and
transmembrane domains of EGF receptor (nucleotides 1-2225) (22) were
fused in-frame with the intracellular domain of short form of Ret
(RET9, nucleotides 2122-end) (23). The fusion was done by generating
an in-frame SalI site as described previously by Santoro
et al. (18). In addition, the carboxyl terminus of the
construct was epitope tagged with the 9 amino acids of influenza
hemagglutinin tag (HA tag).
An XbaI-BamHI fragment containing the first 1347 nucleotides of the EGF receptor was ligated with an 873-base pair
BamHI-SalI fragment to generate the entire
extracellular and transmembrane domains of EGF receptor (plasmid
pSDP10) with an engineered SalI site at the 3 A HindIII-NsiI fragment containing full-length
Ret was obtained by restriction digestion of pSV2c-ret (24). This
fragment was ligated to a 250-base pair NsiI-NotI
fragment containing the HA tag generated by polymerase chain reaction
using pSV2c-ret DNA. The HA tag fragment was generated using
primer 4623 (5 To generate an SalI site in Ret at the junction point,
polymerase chain reaction primers 4499 (5 The final chimeric EGFR/RET construct was then generated by ligating an
SacII-SalI fragment from pSDP10 (5 NIH 3T3 cells were grown in
DMEM containing 10% calf serum and transfected with the appropriate
plasmids using a calcium phosphate precipitation method (26). After 2 days, cells were trypsinized and replated in media containing 400 µg/ml G418. Individual colonies were selected after 2-3 weeks of
growth in G418-containing medium.
For Scatchard analyses,
cells in six-well dishes were incubated for 2 h at 4 °C in DMEM
containing 40 mM HEPES, pH 7.4, 0.1% bovine serum albumin
(HEPES-binding medium) plus 50 pM 125I-EGF and
increasing concentrations of unlabeled EGF. At the end of the
incubation, cells were washed three times in HBSS and were dissolved in
1 ml of 1 M NaOH. The solubilized material was counted for
125I in a For 125I-EGF internalization, cells in six-well dishes were
washed twice with warmed HBSS and then incubated for the indicated period of time with 1 nM 125I-EGF at 37 °C
in HEPES-binding medium. Nonspecific binding was determined in
duplicate wells containing 100 nM unlabeled EGF. At each
time point, half of the cultures were washed three times in HBSS prior
to dissolution in 1 N NaOH and Cells were incubated with 2 or 25 nM EGF for the indicated time at 37 °C. Cultures were
then washed twice at 4 °C with cold HBSS, twice for 2 min with acid
wash buffer, and twice with HBSS. Monolayers were then incubated for
2 h at 4 °C with 50 pM 125I-EGF. Total
125I-EGF binding was determined as indicated above
following three washes with ice-cold HBSS. Nonspecific binding was
determined in duplicate wells containing 100 nM unlabeled
EGF and was subtracted from total binding to yield specific
125I-EGF binding at each time point.
Cells in 60-mm dishes were incubated in DMEM
containing 0.1% bovine serum albumin for 1 h at 37 °C prior to
use. EGF (50 nM) was added directly to the incubation
buffer, and cells were incubated for the indicated times at 37 °C.
At the end of the incubation, cells were washed once in ice-cold HBSS
and lysed by scraping into 300 µl of RIPA buffer (10 mM
Tris, pH 7.2, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1%
deoxycholate, 5 mM EDTA) containing 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 500 µM sodium orthovanadate, and 10 mM
p-nitrophenylphosphate. Lysates were incubated on ice for 10 min with periodic vortexing and then clarified by centrifugation at
12,000 × g for 10 min.
Cells
were plated in six-well dishes and were preloaded with
125I-EGF by incubation for 20 min at 37 °C in the
presence of 1 nM 125I-EGF. Monolayers were
subsequently washed twice in HBSS, twice in acid wash buffer, and twice
in HBSS. All washes were carried out at 4 °C. At time zero prewarmed
DMEM containing 0.1% bovine serum albumin was added to the cultures,
and the cells were shifted to 37 °C. At the indicated times
following the shift to 37 °C, the culture medium was removed. Half
was counted for 125I to determine the total amount of
radiolabel released from the cells. The other half of the medium was
precipitated with 10% trichloroacetic acid to determine the fraction
of released 125I which represented intact, precipitable
125I-EGF.
Aliquots of cell lysates containing
100 µg of protein were subjected to SDS-polyacrylamide gel
electrophoresis on 10% gels and then electrophoretically transferred
to nitrocellulose. The nitrocellulose was blocked using 10% nonfat dry
milk in Tris-buffered saline containing 0.05% Tween 20. Nitrocellulose
membranes were incubated with primary antibody for 1 h at room
temperature, washed, and bands visualized using enhanced
chemiluminescence according to the manufacturer's instructions.
Cells were
plated in D60 plates and grown for 3 days in the presence of standard
growth medium containing 100 µCi of [35S]methionine. At
time zero the labeling medium was removed and replaced with DMEM
containing 0.1% bovine serum albumin in the absence or presence of 50 nM EGF. At the indicated times cultures were extracted with
RIPA buffer and the lysates frozen in liquid nitrogen. When all
cultures had been processed the lysates were thawed and assayed for
protein. Aliquots of cell lysate containing 350 µg of protein were
then immunoprecipitated with an anti-EGF receptor antibody or an
anti-RET antibody. Immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Bands corresponding to the EGF receptor or the EGFR/RET chimera were
excised from the gel and counted for 35S.
Constructs
encoding the wild type EGF receptor or the EGFR/RET chimera were
expressed in NIH 3T3 cells, and stable clones were selected as
described under "Experimental Procedures." Fig. 1
shows Scatchard plots for the binding of 125I-EGF to cells
expressing either the wild type EGF receptor or the EGFR/RET chimera.
Binding of 125I-EGF to the wild type EGF receptor resulted
in a curvilinear Scatchard plot that could be resolved into two classes
of binding sites. The high affinity site accounted for 15-20% of the
total sites and exhibited a Kd of 0.2 nM. The low affinity site exhibited a Kd
of approximately 4 nM. By contrast, binding of
125I-EGF to cells expressing the EGFR/RET chimera generated
a linear Scatchard plot indicative of a single class of sites with a
Kd essentially identical to that of the low affinity
site seen for the wild type EGF receptors. The total number of cell
surface receptors was similar in the clones expressing the wild type
EGF receptor and the EGFR/RET chimera and was approximately 100,000 receptors/cell.
Treatment with EGF led to the enhancement of tyrosine phosphorylation
in cells expressing either the wild type EGF receptor or the EGFR/RET
chimera. As shown in Fig. 2, the EGF receptor and the
EGFR/RET chimera were the principal targets of tyrosine phosphorylation
in the cells. Despite the fact that the two cell lines express similar
numbers of their respective receptors, the level of autophosphorylation
of the EGFR/RET chimeras was substantially lower than the level of
phosphorylation of the wild type EGF receptors. Nonetheless, the
EGF-stimulated phosphorylation of other cellular proteins was largely
the same in the two cell lines. In some experiments, a protein with a
molecular weight of ~65,000 was phosphorylated more strongly in cells
expressing the EGFR/RET chimera than in cells expressing the EGF
receptor (see Fig. 2). This protein was identified as the 62-kDa
Ras-GAP-associated protein by immunoprecipitation (data not shown).
The ability of wild type
EGF receptors and EGFR/RET chimeras to mediate internalization of
125I-EGF was examined next. Cells were incubated in the
presence of 1 nM 125I-EGF at 37 °C for
increasing lengths of time, and the internalized 125I-EGF
was measured as described under "Experimental Procedures." The data
were plotted as the ratio of the internalized ligand to the surface
bound ligand (Fig. 3) (28). The data demonstrate that
both the EGF receptor and the EGFR/RET chimera readily internalized 125I-EGF. However, the rate of internalization of ligand
was 2-fold higher for the chimeric receptor than for the wild type EGF
receptor (0.28 min
The internalization of ligand-receptor complexes leads to receptor
down-regulation if the internalized receptors are targeted to the
lysosomes for degradation. To determine whether ligand internalization
led to the down-regulation of the EGFR/RET chimera, cells expressing
wild type EGF receptors or EGFR/RET chimeras were treated with 2 or 25 nM EGF for increasing periods of time at 37 °C.
Following a wash with low pH buffer to remove surface-bound EGF,
residual cell surface receptors were detected by 125I-EGF
binding (Fig. 4). Pretreatment of cells expressing the
wild type EGF receptor led to the rapid loss of 80-90% of the cell surface 125I-EGF binding. The EGFR/RET chimera also
underwent down-regulation under these conditions. However, the maximal
extent of down-regulation was only half that seen in cells expressing
the wild type EGF receptor. This difference was not the result of
differences in the affinity of the two receptors for EGF as similar
results were seen when subsaturating (2 nM) and saturating
(25 nM) concentrations of EGF were used to down-regulate
the receptor.
Longer incubations of cells with EGF produced similar results.
Treatment of cells with 50 nM EGF for times up to 30 h
led to a progressive loss in binding of 125I-EGF to cell
surface receptors. Scatchard analyses of cells expressing wild type EGF
receptors indicated that treatment with EGF for 24 h led to the
loss of approximately 90% of the cell surface EGF receptors, including
all of the high affinity receptors (Fig. 5A).
Similar analyses of cells expressing the EGFR/RET chimeras demonstrated
that only about 50% of the cell surface receptors were lost after
24 h of EGF treatment (Fig. 5B).
The inefficient down-regulation of the EGFR/RET chimera could be the
result of either enhanced recycling of the chimera to the cell surface
or decreased degradation of the receptor or both. To examine the first
possibility, cells were preloaded with 125I-EGF by
incubation with radioligand for 20 min at 37 °C. Residual cell
surface 125I-EGF was then removed by washing with a low pH
buffer at 4 °C. Cells were shifted to 37 °C, and the
externalization of 125I was followed. As shown in Fig.
6A, the rate of release of 125I
into the culture medium was similar in cells expressing the wild type
EGF receptor and the EGFR/RET chimera. However, in cells expressing the
wild type EGF receptor, approximately 60% of the released material
represented degraded forms of 125I-EGF, whereas for cells
expressing the EGFR/RET chimera, only ~30% of the externalized
material was degraded (Fig. 6B). These data indicate that
125I-EGF is mainly recycled intact by the EGFR/RET chimera,
presumably due to recycling of the receptor to the cell surface and
release of bound ligand.
To determine whether decreased receptor degradation also contributed to
the inefficient down-regulation of the EGFR/RET chimera, the rates of
degradation of wild type EGF receptor and EGFR/RET chimeric receptor
were compared in an [35S]methionine pulse-chase
experiment. As shown in Fig. 7, wild type EGF receptors
exhibited a half-life of approximately 11 h in the absence of EGF.
This decreased to about 4.5 h in the presence of EGF. Thus, ligand
binding enhanced the rate of degradation of the EGF receptor. By
contrast, the addition of ligand had no effect on the rate of
degradation of the EGFR/RET chimera, which exhibited a half-life of
16 h in the absence or presence of EGF.
Western blot analyses of total cellular EGF receptors were consistent
with these data. When cells expressing wild type EGF receptors were
incubated with 50 nM EGF for periods of time ranging from 0 to 24 h, there was a consistent decline in receptor levels throughout the time course (Fig. 8A).
Receptor autophosphorylation paralleled total receptor levels with wild
type EGF receptors exhibiting a slow decline in tyrosine
phosphorylation over the 24-h time period (Fig. 8B). Western
blots using anti-Ret antibodies revealed that the total cellular levels
of the EGFR/RET chimera varied little throughout the 24-h incubation
with EGF, confirming the lack of effect of EGF on the rate of chimeric
receptor degradation. Surprisingly, the EGFR/RET chimeras remained
fully autophosphorylated throughout the entire time course, indicating
that the internalized receptors were active and capable of
signaling.
Prolonged treatment with agonist often leads
not only to receptor down-regulation but also to receptor
desensitization. Receptor desensitization refers to the situation
wherein receptors, although present on the cell surface, are not
functional and are unable to transduce a signal. To determine whether
the residual EGFR/RET chimeric receptors were still functional, cells
expressing wild type EGF receptors or EGFR/RET chimeric receptors were
treated with 25 nM EGF for 20 min to induce down-regulation
of cell surface receptors. After a wash with a low pH buffer,
125I-EGF was added to the cells, and the internalization of
this ligand was assessed after 5 min at 37 °C. As shown in Fig.
9, pretreatment with EGF of cells expressing the wild
type EGF receptor led to a 90% loss in the ability of the receptor to
internalize 125I-EGF, presumably due to the internalization
and down-regulation of the bulk of the cell surface receptors. Although
the cells expressing the EGFR/RET chimera exhibited approximately a
40-50% loss in cell surface receptor number (Fig. 4), their ability
to internalize 125I-EGF subsequently was reduced by only
~25% compared with controls. These results indicate that the
residual cell surface EGFR/RET chimeras remain functional after
down-regulation has occurred.
PMA is a tumor-promoting phorbol ester that
stimulates the activity of protein kinase C and leads to the
phosphorylation of threonine residues within the EGF receptor (29).
Treatment of cells with PMA results in the loss of cell surface EGF
receptors and an inhibition of EGF-stimulated tyrosine kinase activity
(30-33). To determine whether the Ret tyrosine kinase was also subject to regulation by a protein kinase C-mediated pathway, cells expressing wild type EGF receptors or EGFR/RET chimeras were treated for 30 min at
37 °C in the absence or presence of 100 nM PMA and
analyzed for 125I-EGF binding. Treatment with PMA resulted
in the loss of 90% of 125I-EGF binding to the wild type
EGF receptor. Scatchard analyses indicated that the decrease in binding
was due to the loss of all high affinity and most low affinity sites as
well a reduction in the affinity of the low affinity sites for EGF from
3.8 ± 0.2 nM to approximately 15 ± 3 nM (n = 2). The effect of PMA on the binding of 125I-EGF to cells expressing EGFR/RET chimeras
was limited and consisted solely of a small decrease in the affinity of
the single class of binding sites for EGF from 5 ± 1 nM to 8 ± 2 nM (n = 2)
(data not shown).
Fig. 10 shows the dose-response curves to EGF for the
stimulation of receptor autophosphorylation in cells treated with or without 100 nM PMA. In cells expressing the wild type EGF
receptor, prior treatment with PMA significantly blocked EGF-stimulated autophosphorylation at every level of EGF examined. By contrast, pretreatment with PMA had little effect on the ability of EGF to
stimulate autophosphorylation in cells expressing the EGFR/RET chimera.
Thus, the activity of the ret tyrosine kinase does not appear to be
modulated via a protein kinase C-dependent mechanism.
Previous studies of EGFR/RET chimeras have demonstrated that the
chimeras respond to EGF and mediate the activation of a variety of
downstream signaling pathways including the activation of phospholipase C, Ras, and MAP kinase (18, 19). The studies reported here extend these
findings by providing insight into the function and regulation of the
EGFR/RET chimera.
Many growth factor receptor kinases, including the EGF receptor,
exhibit high and low affinity binding sites for their ligands. Although
the presence of two classes of EGF binding sites was readily
demonstrable in cells transfected with the wild type EGF receptor,
cells expressing the EGFR/RET chimera consistently showed only a single
class of low affinity binding sites for EGF. In the case of the EGF
receptor, the presence of two classes of binding sites has been
attributed to the ability of the receptor to dimerize (34-36). Dimer
formation occurs even in the isolated, soluble extracellular domain of
the EGF receptor, suggesting that this domain alone is capable of
inducing receptor dimerization (37). The fact that the EGFR/RET chimera
that contains the extracellular and transmembrane domains of the EGF
receptor fails to exhibit high affinity ligand binding sites implicates
the cytosolic domain in some aspect of high affinity ligand binding.
Experiments demonstrating a loss of high affinity binding in EGF
receptors with mutations in the intracellular domain are consistent
with a role for the cytosolic portion of the receptor in high affinity
ligand binding (38). It is possible that high affinity ligand binding
is stabilized by interactions between the intracellular domains of EGF
receptor monomers that are lacking in the Ret tyrosine kinase. Glial
cell-derived neurotrophic factor (GDNF), the Ret ligand, binds with
only low affinity to the Ret tyrosine kinase. High affinity GDNF
binding requires the presence of an auxiliary protein, termed the
GDNF- Despite the fact that the EGFR/RET chimera exhibited only low affinity
EGF binding, the receptor was able to transduce a signal in NIH 3T3
cells. In particular, EGF stimulated the autophosphorylation of
EGFR/RET. However, although binding studies indicated that the clones
used in these experiments expressed similar numbers of EGF receptors or
EGFR/RET chimeras, the level of autophosphorylation of the EGFR/RET
chimera was substantially lower than that of the EGF receptor. This
difference in autophosphorylation could be due to the presence of fewer
sites of autophosphorylation in Ret than in the EGF receptor or could
be the result of a lower extent of phosphorylation overall. Liu
et al. (39) recently reported findings that suggest that Ret
contains five or six sites of autophosphorylation, a number similar to
that found in the EGF receptor. Thus, the lower level of
autophosphorylation observed in the EGFR/RET chimera is likely to be
due to a reduced overall level of phosphorylation of the chimera. This
does not appear to be the result of a decrease in the intrinsic
tyrosine kinase activity of EGFR/RET, as the phosphorylation of other
cellular proteins was similar to that seen in cells expressing the wild
type EGF receptor.
EGFR/RET internalized 125I-EGF at a rate that was
approximately 2-fold higher than the rate of ligand internalization by
wild type EGF receptors. Internalization of receptors appears to be mediated by cytoplasmic sequences that promote clustering of receptors in coated pits and interaction with clathrin adapter proteins (40, 41).
Sequences containing an Asn-Pro-Xaa-Tyr motif have been shown to
mediate receptor internalization (42); however, no such sequence is
found in RET. The motif, Tyr-Xaa-Xaa-hydrophobic, where Xaa stands for
any amino acid, also appears to mediate receptor internalization (43).
The sequence Tyr1015-Leu-Asp-Leu in the RET cytoplasmic
domain fulfills the criteria for an internalization signal. Whether
this sequence serves this function remains to be determined.
Although the EGFR/RET chimera is internalized more rapidly than the EGF
receptor, it is down-regulated less extensively. Scatchard analyses
indicated that 90% of cell surface EGF receptors were lost after a
24-h treatment with EGF, whereas only 50% of cell surface EGFR/RET
chimeras were lost. The observation that EGF induces the
down-regulation of 50% of the cell surface EGFR/RET receptors suggests
that ligand binding promotes internalization of the chimera. However,
pulse-chase studies as well as Western blotting demonstrated that EGF
did not enhance the rate of degradation of the EGFR/RET chimera. This
contrasts markedly with the findings observed for the EGF receptor for
which ligand promoted both receptor internalization and receptor
degradation. These findings suggest that Ret may lack an effective
lysosomal targeting signal and demonstrate a marked difference in the
intracellular trafficking of the EGFR/RET chimera and the wild type EGF
receptor.
Perhaps as a result of reduced lysosomal targeting, the EGFR/RET
chimera appeared to be recycled to the plasma membrane more extensively
than the EGF receptor. Approximately two-thirds of the internalized
125I-EGF was released intact from cells expressing EGFR/RET
chimeras, whereas cells expressing wild type EGF receptors released
only one-third of their internalized 125I-EGF intact.
Assuming that the release of intact 125I-EGF is due to
recycling of the receptor to the plasma membrane, this suggests that
EGFR/RET is preferentially recycled to the plasma membrane, whereas the
EGF receptor is preferentially degraded. The combination of reduced
receptor degradation and enhanced receptor recycling apparently gives
rise to the limited down-regulation observed in cells expressing the
EGFR/RET chimera.
Interestingly, the internalized EGFR/RET chimeras retained their levels
of tyrosine phosphorylation throughout a 24-h incubation with EGF.
Since approximately half of the chimeras are present in an
intracellular compartment at the 24-h time point, this suggests that
the internalized receptors remain active and capable of signaling. Furthermore, the 50% of the receptors remaining on the cell surface appeared to be functional, at least in terms of ligand internalization. These observations suggest that the maintenance of receptor function under conditions that typically lead to a loss of receptor activity is
an intrinsic feature of the Ret tyrosine kinase.
Protein kinase C has been shown to phosphorylate the EGF receptor
leading to decreases in EGF binding and receptor tyrosine kinase
activity. Although treatment of cells expressing the wild type EGF
receptor with PMA to activate protein kinase C clearly led to a marked
decrease in 125I-EGF binding and receptor
autophosphorylation, cells expressing the EGFR/RET chimera showed
little response to PMA. Thus, the Ret tyrosine kinase does not appear
to be regulated by a protein kinase C-dependent mechanism.
The observation that the activity and function of the EGFR/RET chimera
are not diminished markedly by either down-regulation with EGF or
activation of protein kinase C indicates that Ret is unresponsive to
two major forms of regulation designed to suppress receptor-mediated
signaling. This suggests that physiologically it may be important for
cells to maintain a constant sensitivity to Ret ligand and to prolong
the lifetime of the activated form of the Ret tyrosine kinase.
Recently the Ret ligand has been identified as GDNF (13-17). GDNF has
been shown to promote the survival of dopaminergic and motor
neurons following axotomy or treatment with dopaminergic neurotoxins
(44-48). GDNF undergoes receptor-mediated retrograde axonal transport
(49), suggesting that it is a target-derived neurotrophic factor. In
such systems the growth factor-receptor complex is internalized at the
nerve terminal and subsequently transported to the cell body, a process
that can take a significant amount of time. If the goal of this process
is to deliver a signaling-competent, ligand-receptor complex to the
cell body, then the ability of Ret to internalize ligand rapidly,
maintain its activated state, and avoid degradation in the lysosomes is
consistent with a role for this receptor in mediating the effects of a
target-derived neurotrophic factor.
These studies have identified unique functional properties of the
EGFR/RET chimeric receptor. The absence of ligand-stimulated receptor
degradation as well as the persistent autophosphorylation of the
chimera indicate that Ret is not subject to traditional mechanisms used
to regulate receptor function. These characteristics, which differ from
those of the EGF receptor, may be relevant to the physiological role of
the Ret tyrosine kinase in growth and development.
Surgery,
combined in
ransfection) gene encodes a receptor tyrosine kinase
homolog involved in innervation of the gut and renal development. A
chimeric epidermal growth factor receptor (EGFR)/RET receptor was
constructed which contained the extracellular and transmembrane domains
of the EGF receptor fused to the intracellular domain of
RET. This construct was expressed in NIH 3T3 cells, and
the functional properties of the receptor were characterized and
compared with those of the wild type EGF receptor. Whereas the EGF
receptor exhibited both high and low affinity binding sites for
125I-EGF, the EGFR/RET chimera exhibited only low affinity
binding of 125I-EGF. The chimera was able to internalize
EGF more rapidly than the wild type EGF receptor and recycled to the
cell surface at twice the rate of the EGF receptor. Pulse-chase
experiments indicated that EGF stimulated the degradation of the wild
type EGF receptor but had no effect on the rate of degradation of the
EGFR/RET receptor. The combination of increased recycling and decreased
degradation resulted in the relatively inefficient down-regulation of
the EGFR/RET chimera. Incubation of cells expressing the wild type EGF
receptor with phorbol 12-myristate 13-acetate led to a reduction in
125I-EGF binding and a loss in EGF-stimulated tyrosine
phosphorylation. However, phorbol 12-myristate 13-acetate treatment had
only a limited effect on EGF binding and EGF-stimulated tyrosine kinase activity in cells expressing EGFR/RET chimeras. These findings suggest
that the ret tyrosine kinase is not regulated by many of the common
mechanisms used to terminate signaling via growth factor receptors.
Such persistent activation of the Ret tyrosine kinase may be relevant
to the physiological function of Ret in cells that normally express
this growth factor receptor.
combined in
ransfection to generate a fusion protein that induced the
transformation of NIH 3T3 cells (3). Subsequent studies have shown
that activating mutations in the RET gene are responsible
for several dominantly inherited human neoplasias including multiple
endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, and
familial medullary thyroid carcinoma (4-8). These conditions are all
characterized by medullary thyroid carcinoma with variable involvement
of other endocrine tissues such as the adrenal gland and the
parathyroid gland.
end. The
873-base pair BamHI-SalI fragment was generated
by polymerase chain reaction using primers corresponding to nucleotides
1331-1350 of the EGF receptor and a primer sequence (5
-CCTCTCCTGCAGCA
GCAGCGTGCGC-3
) to create the
SalI site (underlined) at nucleotide 2219 (amino acid 657)
of the EGF receptor sequence.
-TCCC
TTACTAGATTC
TAGCACCGCTGTCCCCTCTGC-3
), which contains an NsiI site (underlined) and inserts
the nine amino acids of the HA tag (underlined) immediately before the stop codon of RET9, and primer 4646 (5
-CCGG
CCAACTCGCGAGGGGATCGAG-3
), which introduces
an NotI site (underlined) and is in the 5
end of the
neomycin gene of pSV2c-ret. This construct was placed in pSK1
Bluescript to generate the full-length epitope-tagged form of RET.
-AT
ACTGCTACCACAAGTTTGCC-3
, nucleotides 2122-2142
of RET) and 4500 (antisense nucleotides 2318-2298) were used to
amplify a ~200-base pair fragment that contained an engineered
SalI site. The polymerase chain reaction product was
digested with SalI and BamHI. This linker
fragment was then ligated to a BamHI-NotI
fragment (from epitope-tagged RET) and
SalI-NotI-digested pSK1 Bluescript in a
three-fragment ligation to generate plasmid pSDP18.
end of the
EGF receptor) with the SalI-NotI fragment from
pSDP18 (3
end of epitope-tagged RET) and
SacII-NotI-digested pM2-HA vector (25) in a
three-fragment ligation to generate plasmid pSDP24.
counter. Data were analyzed using the LIGAND
computer program (27).
counting. This provided
a measure of total cell-associated 125I-EGF. The other half
of the cultures were washed twice for 2 min in acid wash buffer (50 mM glycine, 100 mM NaCl, pH 3.0) prior to
dissolution and
counting. This quantitated the internalized radioligand. For In/Sur plots, the surface 125I-EGF was
calculated by subtracting the internalized 125I-EGF from
the total cell-associated 125I-EGF.
Ligand Binding and Tyrosine Phosphorylation
Fig. 1.
Scatchard plot for the binding of
125I-EGF to cells expressing wild type EGF receptors or
EGFR/RET chimeras. Cells were plated in 35-mm dishes and cultured
for 2 days prior to use. 125I-EGF binding and Scatchard
analyses were carried out as described under "Experimental
Procedures." Points represent the mean of triplicate
determinations.
[View Larger Version of this Image (20K GIF file)]
Fig. 2.
EGF-stimulated tyrosine phosphorylation in
cells expressing wild type EGF receptors or EGFR/RET chimeras.
Parental NIH 3T3 cells or NIH 3T3 cells expressing the wild type EGF
receptor or the EGFR/RET chimera were treated with 50 nM
EGF for the indicated times. Lysates were prepared in RIPA buffer and
analyzed by SDS-polyacrylamide gel electrophoresis followed by Western
blotting using an anti-phosphotyrosine antibody. Arrowheads
indicate the location of the EGF receptor (left arrowhead)
or the EGFR/RET chimera (right arrowhead). Molecular masses
of marker proteins (in kDa) are indicated on the right and
left of the panel.
[View Larger Version of this Image (53K GIF file)]
1 versus 0.14 min
1 for the chimera and the EGF receptor,
respectively).
Fig. 3.
Internalization of 125I-EGF by
the wild type EGF receptor or the EGFR/RET chimera. Cells were
plated in 35-mm dishes and cultured for 2 days prior to use. Monolayers
were washed twice in warm HBSS and transferred to warm HEPES-binding
medium containing 1 nM 125I-EGF. Cells were
incubated at 37 °C for the indicated times. Total cell-associated
125I-EGF and internalized 125I-EGF were then
determined as described under "Experimental Procedures." Cell
surface counts were calculated by subtracting the counts internalized
from the total cell-associated counts. The results are reported as the
ratio of the internalized counts to the counts present on the cell
surface at the indicated time. Points represent the mean of
triplicate determinations.
[View Larger Version of this Image (16K GIF file)]
Fig. 4.
Short term down-regulation of the EGF
receptor and the EGFR/RET chimeric receptor. Cells grown on 35-mm
plates were treated with 2 or 25 nM EGF at 37 °C for the
indicated time. Monolayers were subsequently washed with cold HBSS and
acid wash buffer to remove cell surface EGF. Binding of 50 pM 125I-EGF was then determined as outlined
under "Experimental Procedures." Results represent the mean of
triplicate determinations.
[View Larger Version of this Image (24K GIF file)]
Fig. 5.
Scatchard analysis of cells expressing EGF
receptors or EGFR/RET chimeric receptors after a 24-h incubation with
EGF. Cells expressing wild type EGF receptors or EGFR/RET chimeras were plated on 35-mm plates and grown to confluence. Cultures were then
incubated for 24 h in the absence (open symbols) or presence (closed symbols) of 50 nM EGF.
Monolayers were washed with cold HBSS and acid wash buffer to remove
cell surface EGF. 125I-EGF binding and Scatchard analyses
were then performed as outlined under "Experimental Procedures."
Values represent the mean of triplicate determinations and have been
corrected for the ~30% increase in cell number which occurred in
each cell line in the presence of EGF. Cells expressing wild type EGF
receptors grew to a density of 4 × 105 cells/well in
the presence of EGF. Cells expressing EGFR/RET chimeras grew to a
density of 9 × 105 cells/well in the presence of
EGF.
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
Recycling and degradation of
125I-EGF. Cells expressing wild type EGF receptors or
EGFR/RET chimeric receptors were preloaded with 125I-EGF as
described under "Experimental Procedures." At the indicated times
after shift of the cultures to 37 °C, the culture medium was
collected and an aliquot counted for 125I to determine the
fraction of internalized 125I which had been released into
the culture medium (panel A). A second aliquot was subjected
to precipitation with 10% trichloroacetic acid, and the
trichloroacetic acid-soluble supernatant was counted to determine the
fraction of released 125I which represented degraded forms
of 125I-EGF (panel B). Points represent the
mean ± S.D. of triplicate determinations.
[View Larger Version of this Image (25K GIF file)]
Fig. 7.
Degradation of EGF receptors and EGFR/RET
chimeric receptors. Cells expressing wild type EGF receptors or
EGFR/RET chimeric receptors were metabolically labeled with
[35S]methionine. At time zero the labeling medium was
replaced with standard culture medium containing vehicle (open
circles) or 50 nM EGF (closed circles). At
the indicated times, cells were lysed in RIPA buffer and
immunoprecipitated using and anti-EGFR antibody or an anti-ret
antibody. Immunoprecipitates were run on 10% SDS-polyacrylamide gels
and subjected to autoradiography. Panel A, autoradiographs of the SDS gel. Panel B, bands representing the EGF receptor
or the EGFR/RET chimeric receptor were excised from the gel and counted for 35S.
[View Larger Version of this Image (23K GIF file)]
Fig. 8.
Receptor levels and autophosphorylation
during long term treatment with EGF. Parental cells or cells
expressing wild type EGF receptors or EGFR/RET chimeric receptors were
grown for the indicated times in the presence of 50 nM EGF.
Lysates were then prepared and subjected to SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to nitrocellulose and
analyzed by Western blotting using an anti-EGF receptor or anti-Ret
antibody (panel A) or an anti-phosphotyrosine antibody
(panel B).
[View Larger Version of this Image (49K GIF file)]
Fig. 9.
Down-regulation of 125I-EGF
internalization by wild type EGF receptors and EGFR/RET chimeric
receptors. Cells expressing EGF receptors or EGFR/RET chimeras
were incubated in the absence (Control) or presence
(Down Regulated) of 25 nM EGF for 20 min at
37 °C. Monolayers were washed with cold HBSS and acid wash buffer to
remove cell surface EGF. Cells were then incubated in HEPES-binding
medium at 37 °C for 5 min in the presence of 1 nM 125I-EGF. Total cell-associated 125I-EGF and
internalized 125I-EGF were determined as described under
"Experimental Procedures." Results represent the mean ± S.D.
of triplicate determinations.
[View Larger Version of this Image (33K GIF file)]
Fig. 10.
Effect of PMA on receptor
autophosphorylation. Cells expressing wild type EGF receptors or
EGFR/RET chimeric receptors were treated with 100 nM PMA
for 30 min at 37 °C. Cultures were then incubated with increasing
concentrations of EGF for 5 min at 37 °C, and lysates were prepared.
Proteins were separated by SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose. Tyrosine phosphorylation was visualized
by Western blotting with an anti-phosphotyrosine antibody.
[View Larger Version of this Image (28K GIF file)]
receptor (15, 16). The GDNF-
receptor is a
glycosylphosphatidylinositol-linked protein with no intracellular
domain. Thus, it is unlikely that it interacts directly with the
tyrosine kinase domain of Ret. However, it is possible, that the
GDNF-
receptor aids dimer formation, promoting interaction of the
cytosolic domains of Ret and stabilizing high affinity binding.
*
This work was supported in part by National Institutes of
Health Grant P01CA5352 (to H. D.-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: Dept. of
Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid, Box 8231, St. Louis, MO 63110. Fax:
314-362-7183.
1
The abbreviations used are: EGF, epidermal
growth factor; EGFR, EGF receptor; MAP, mitogen-activated protein; HA,
hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; HBSS,
Hanks' balanced salt solution; PMA, phorbol 12-myristate 13-acetate;
GDNF, glial cell-derived neurotrophic factor.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.