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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 898-906
Analysis of c-kit Receptor Dimerization by Fluorescence
Resonance Energy Transfer
By
Virginia C. Broudy,
Nancy L. Lin,
Hans-Jörg Bühring,
Norio Komatsu, and
Terrance J. Kavanagh
From the Departments of Medicine and Environmental Health, University
of Washington, Seattle; the Department of Medicine, University of
Tübingen, Tübingen, Germany; and the Department of
Medicine, Jichi Medical School, Tochigi, Japan.
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ABSTRACT |
Stem cell factor (SCF) binding to the c-kit receptor
triggers homodimerization and intermolecular tyrosine phosphorylation of the c-kit receptor, thus initiating signal transduction.
Receptor dimerization is a critical early step in this process. Prior
biochemical studies of c-kit receptor dimerization have mainly
used affinity cross-linking techniques, which are beset with problems
including low efficiency of cross-linking and the usual requirement for radiolabeled SCF to detect the cross-linked complex. We used the fluorescence resonance energy transfer (FRET) technique to examine the
effects of SCF and other hematopoietic cytokines on c-kit receptor dimerization. The nonneutralizing anti-c-kit receptor monoclonal antibody 104D2 was directly conjugated to fluorescein isothiocyanate (FITC) or to the carbocyanine dye Cy3 and used to label
cytokine-responsive human hematopoietic cell lines. The ability of SCF
to induce c-kit receptor dimerization was assessed by flow
cytometric analysis of FRET between the donor fluorochrome FITC and the
acceptor fluorochrome Cy3. SCF induced a dose-dependent increase in
c-kit receptor dimerization that correlated well with the
concentrations of SCF required to stimulate cell proliferation. Receptor dimerization was detectable within 3 minutes after the addition of SCF and was maximal 30 minutes after the addition of SCF.
Confocal microscopy showed redistribution of the c-kit receptor
(from a diffuse distribution on the cell surface to "caps" at one
end of the cell) within 3 minutes after SCF addition, followed by
receptor internalization. Reappearance of the c-kit receptor on
the cell surface required new protein synthesis, suggesting that the
c-kit receptor is not recycled to the cell surface after internalization. Finally, erythropoietin (Epo), but not the
structurally and functionally related cytokine thrombopoietin (Tpo),
stimulated c-kit receptor dimerization detectable by FRET, and
tyrosine phosphorylation of the c-kit receptor. These results
suggest that exposure to Epo can activate the c-kit receptor
and provide further evidence for cross-talk between the Epo and
c-kit receptors in human hematopoietic cell lines. Studies with
progeny of burst-forming unit-erythroid (BFU-E) suggest
that the FRET technique is sufficiently sensitive to detect
c-kit receptor dimerization on normal human hematopoietic cells.
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INTRODUCTION |
STEM CELL FACTOR (SCF) is a growth factor
that stimulates the survival, proliferation, and differentiation of
hematopoietic cells.1 SCF also promotes mast cell
production and function and plays a role in the development of
melanocytes, germ cells, and intestinal pacemaker cells. Endothelial
cells, fibroblasts, and certain epithelial cells constitutively produce
soluble and transmembrane forms of SCF, and the soluble form of SCF
circulates in the blood as a noncovalently associated
dimer.2
SCF initiates its biologic effects by binding to the c-kit
receptor,3,4 a member of the type III receptor tyrosine
kinase family.5 The c-kit receptor is encoded at
the murine W locus. The dominant negative effect of mutations
at the W locus that ablate or diminish c-kit receptor
tyrosine kinase activity yet permit cell surface expression of the
impuissant protein first suggested that the c-kit receptor
might be activated by homodimerization and
autophosphorylation.6-8 Enforced expression of
kinase-deficient W42 c-kit receptor in
transgenic mice recapitulated some of the phenotypic abnormalities
found in mice with naturally occurring mutations at the W locus
and provided support for this model of c-kit receptor activation.9
Dimerization of the c-kit receptor in the presence of SCF has
been directly demonstrated by affinity cross-linking
techniques10-12 and by biophysical
methods.13,14 The current concept is that binding of
dimeric SCF triggers c-kit receptor homodimerization and
intermolecular tyrosine phosphorylation of the receptor, creating a
molecular scaffolding that contains docking sites for SH2-containing signal transduction molecules.15,16 Thus, c-kit
receptor dimerization is a key initial step in the SCF signal
transduction process.
The biochemical10-12 and biophysical13,14
studies of c-kit receptor dimerization do not provide an
optimal way to examine a dynamic process such as assembly of receptor
subunits on an intact cell membrane. Fluorescence resonance energy
transfer (FRET) between a donor fluorochrome and an acceptor
fluorochrome is exquisitely dependent on the distance separating the
two fluorochromes, and thus can serve as a "molecular
ruler"17,18 within the 10 to 75 angstrom range. The FRET
technique has been used to investigate assembly of the T-cell antigen
receptor complex,19,20 to demonstrate interleukin-1 (IL-1)
receptor dimerization after IL-1 binding,21 and to study
epidermal growth factor (EGF) receptor oligomerization,22,23 among other uses.24
We wished to define in greater detail the kinetics of c-kit
receptor dimerization in intact cells. Because of the importance of
both SCF and erythropoietin (Epo) for normal erythropoiesis and the
accumulating evidence for cross-talk between the c-kit and Epo
receptors,25-27 we also wished to determine whether
exposure to Epo could induce c-kit receptor dimerization. To
achieve these goals, we used the FRET technique to permit assessment of
c-kit receptor dimerization without the use of affinity
cross-linking. We initially showed that SCF-induced c-kit
receptor dimerization can be detected by FRET between monoclonal
anti-c-kit receptor antibodies labeled with the donor
fluorochrome fluorescein isothiocyanate (FITC) and the acceptor
fluorochrome Cy3. Dimerization of the c-kit receptor was
detectable within 3 minutes of SCF addition. We then applied this
technique to determine whether other cytokines could induce
c-kit receptor dimerization. The results suggest that Epo, but
not the structurally and functionally similar cytokine thrombopoietin
(Tpo), or the myeloid cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), can activate the c-kit
receptor in cytokine-responsive cell lines. Minutes after the addition of SCF, redistribution of the c-kit receptor into "caps"
on the cell surface occurs. Moreover, the c-kit receptor
remains dimerized after internalization of the ligand-receptor complex
and does not recycle to the cell surface.
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MATERIALS AND METHODS |
Cells.
The SCF-responsive human hematopoietic cell line MO7e28,29
was maintained in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS, Hyclone, Logan, UT) and recombinant human GM-CSF (3 ng/mL; a gift from
Dr Kenneth Kaushansky, University of Washington, Seattle). The UT-7/Epo
cell line30 was maintained in IMDM supplemented with 10%
FCS and 1 U/mL recombinant human Epo.31 The UT-7/Tpo cell
line32 was maintained in IMDM supplemented with 10% FCS and 10 ng/mL recombinant human Tpo (obtained from Zymogenetics, Inc,
Seattle, WA). Normal human burst-forming unit-erythroid (BFU-E) progeny
were obtained as previously described.33,34
Nonhemoglobinized BFU-E progeny were plucked on day 10 of culture and
were incubated in IMDM supplemented with 5% FCS plus 1 ng/mL
recombinant human IL-3 (obtained from Dr Kenneth Kaushansky) for 3 hours at 37°C to permit internalization and degradation of
surface-bound SCF acquired during culture,33 before use for
FRET experiments.
FRET technique.
The 104D2 anti-c-kit receptor monoclonal antibody (IgG1) was
generated by immunizing a Balb/C mouse with the MOLM-1 cell line and
screening the hybridomas for ability to specifically bind to NIH/3T3
cells engineered to express human c-kit receptor, but not to
control NIH/3T3 cells.35,36 This antibody does not block binding of 125I-SCF to the c-kit receptor
(Table 1). Purified 104D2 (1 mg) was
directly conjugated to FITC (Molecular Probes, Inc, Eugene, OR) as
recommended by the manufacturer. A separate aliquot of purified 104D2
(1 mg) was directly conjugated to Cy3 using a FluoroLink Cy3 Reactive
Dye 5-Pack (Amersham Life Science, Arlington Heights, IL). Both
conjugations were performed at 3 different molar ratios of dye to
protein. The final coupling ratio was determined as previously
described.21 A dye to protein molar ratio of approximately 2.5 to 1 resulted in retention of the ability of 104D2 to recognize the
c-kit receptor on MO7e cells and in sufficient fluorescence intensity for use of the FRET technique.
To assess c-kit receptor dimerization, the SCF responsive cell
lines MO7e, UT-7/Epo, or UT-7/Tpo were labeled with a mixture of
104D2-FITC and 104D2-Cy3 (0.1 to 0.3 µg/mL of each conjugate) for 1 hour at 4°C, then incubated with or without various concentrations of recombinant human SCF (expressed in E. coli, provided by
Amgen, Inc, Thousand Oaks, CA) for 3 minutes to 1 hour at 37°C. The
cells were then fixed in 1% paraformaldehyde and analyzed in a Coulter Epics Elite flow cytometer (Coulter, Miami, FL) with 488 nm excitation. FITC fluorescence emission was detected at 505 to 545 nm, and Cy3
fluorescence emission was detected above 590 nm. Because some FITC
fluorescence can be detected at the wavelength used to detect Cy3
fluorescence,21 the fraction of FITC fluorescence that
crosses into the Cy3 window was electronically compensated.
Fluorescence was measured with linear signal amplification. To
determine whether other hematopoietic cytokines could induce
c-kit receptor dimerization, the cells labeled with 104D2-FITC
plus 104D2-Cy3 were incubated with Epo (4 U/mL), IL-3 (2.5 ng/mL),
GM-CSF (2.5 ng/mL), or Tpo (50 ng/mL) for 1 hour at 37°C, and
c-kit receptor dimerization was assessed by FRET as described
above. To examine the effect of reduced temperature on c-kit
receptor dimerization, MO7e cells labeled with 104D2-FITC plus
104D2-Cy3 at 4°C were incubated without or with SCF at either
37°C or 4°C before analysis.
The FRET data were analyzed as previously described.21 In
brief, the mean fluorescence intensity of the 104D2-FITC and of the
104D2-Cy3 bound to the cells was determined by flow cytometric analysis. Each experiment included a sample of cells labeled with 104D2-FITC plus 104D2-Cy3 in the presence of a 20-fold molar excess of
unconjugated 104D2. The equation used to calculate the acceptor (Cy3)
to donor (FITC) ratio is: Acceptor/Donor Ratio=mean fluorescence intensity of 104D2-Cy3 minus mean fluorescence intensity of (104D2-Cy3 plus unconjugated 104D2)/mean fluorescence intensity of 104D2-FITC minus mean fluorescence intensity of (104D2-FITC plus unconjugated 104D2).
Analysis of c-kit receptor surface display.
The kinetics of SCF-induced c-kit receptor internalization and
of c-kit receptor reappearance at the cell surface were
analyzed by flow cytometry. MO7e cells were cultured overnight without GM-CSF in IMDM supplemented with 5% FCS and 0.5% bovine serum albumin
(BSA; Intergen, Purchase, NY), then incubated with SCF (100 ng/mL) for
1 hour at 37°C. After the incubation with SCF, the cells were
washed three times to remove the SCF and resuspended in IMDM
supplemented with 5% FCS and 0.5% BSA. At various time points (0 to 4 hours), an aliquot of cells was removed, and cell surface c-kit
receptor display was detected by labeling the cells with 104D2 (2 µg/mL) followed by goat antimouse IgG-PE (Jackson ImmunoResearch,
West Grove, PA) and flow cytometric analysis. In parallel, to determine
whether reappearance of the c-kit receptor at the cell surface
required new protein synthesis, MO7e cells were incubated with SCF (100 ng/mL) plus cycloheximide (10 µg/mL; Sigma Chemical Co, St Louis, MO)
for 1 hour at 37°C. The cells were washed three times as described
above and resuspended in IMDM supplemented with 5% FCS, 0.5% BSA, and
cycloheximide (10 µg/mL). Aliquots of cells were removed at various
time points (0 to 4 hours), and cell surface c-kit receptor
display was analyzed as described above.
Analysis of c-kit receptor phosphorylation.
UT-7/Epo or UT-7/Tpo cells were washed twice and incubated overnight at
37°C in IMDM supplemented with 5% FCS in the absence of exogenous
growth factors. The cells were then exposed to individual growth
factors in IMDM (SCF 150 ng/mL, Epo 2.5 U/mL, GM-CSF 5 ng/mL, or Tpo 25 ng/mL) for 10 minutes at 37°C, then lysed by rocking for 20 minutes
at 4°C in a solution consisting of 20 mmol/L Tris, 150 mmol/L NaCl,
10 mmol/L EDTA, 10% glycerol, 1% Triton X-100, 1.5 mmol/L
MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 200 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µmol/L pepstatin, 100 µmol/L NaF, and 2 mmol/L Na orthovanadate, as previously
described.37 Triton X-100, leupeptin, and aprotinin were
obtained from Boehringer Mannheim (Indianapolis, IN). The other
chemicals were obtained from Sigma. The cell lysates were centrifuged
for 5 minutes at 10,000g to remove insoluble debris, and
solubilized cellular proteins were precleared by precipitation with
protein A-sepharose beads (Pharmacia LKB, Piscataway, NJ). The
c-kit receptor was immunoprecipitated by incubating with
purified SR-1 monoclonal antibody (4 µg/mL)38 for 3 hours
at 4°C, followed by the addition of protein A-sepharose beads (1 hour at 4°C). The immunoprecipitates were washed once with 20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 0.1% Triton X-100, 10%
glycerol, then resuspended in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer containing 50 mmol/L dithiothreitol,12 boiled for 2 minutes, and analyzed on a
7% SDS polyacrylamide gel. The proteins were transferred to
nitrocellulose membranes (Bio-Rad, Richmond, CA), and
tyrosine phosphorylated proteins were detected by Western blotting with
the 4G10 antiphosphotyrosine monoclonal antibody (Upstate
Biotechnology, Inc, Lake Placid, NY) and the enhanced chemiluminescence
system (Amersham).
Confocal microscopy.
Confocal microscopy was used to determine whether SCF would alter the
localization of the c-kit receptor on the cell surface, and to
assess the time course of c-kit receptor internalization. MO7e
cells growing in GM-CSF were washed free of GM-CSF and resuspended in
IMDM supplemented with 0.5% BSA and SCF (200 ng/mL). Aliquots of the
cells were removed after 3 minutes, 10 minutes, or 30 minutes at
37°C. The cells were fixed in 0.5% paraformaldehyde and one half
of each aliquot of cells was permeabilized with 0.05% Triton X-100 (to
permit detection of intracellular c-kit receptor). All of the
samples were labeled with 104D2 (5 µg/mL) for 45 minutes at 4°C,
washed, and then incubated with goat antimouse IgG-FITC for 45 minutes
at 4°C. Just before confocal microscopy, the cells were incubated
with propidium iodide (7.5 µg/mL; Molecular Probes, Inc) in 0.1%
citrate plus 0.1% Triton X-100 to stain the nuclei. Surface
fluorescence and intracellular fluorescence was analyzed with an ACAS
Ultima Laser Cytometer (Meridian Instruments, Okemos, MI) as previously
described.39 Fluorescence excitation was at 488 nm and FITC
emission was detected with a 530/20 band pass filter. Propidium iodide
emission was detected with a 605 long pass filter. A 60× oil
immersion objective (numerical aperture 1.3) was used for scanning
cells with a step (pixel) size of 0.2 µm in the xy plane and a step
size of 0.4 µm for serial optical sections in the z axis. The pinhole
setting was 225 µm, which yielded a theoretical optical thickness
(full width at half maximum) of approximately 1 µm.
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RESULTS |
The FRET technique can be used to detect c-kit receptor
dimerization on a human hematopoietic cell line
(Table 2). MO7e cells were labeled with the
nonneutralizing anti-c-kit receptor monoclonal antibody 104D2
that had been directly conjugated to either FITC or to Cy3. After
incubation with SCF at 37°C, the mean fluorescence intensity of the
donor fluorochrome (FITC) decreased, while the mean fluorescence
intensity of the acceptor fluorochrome (Cy3) increased, in comparison
to incubation without SCF, indicating that FRET occurred between the
two labeled antibodies (Table 2). Because the distance at which 50% of
the energy is transferred (Ro) is 55 angstroms for the
FITC-Cy3 pair of fluorochromes,21 we interpret these
results to mean that a portion of the c-kit receptor monomers
must have dimerized in the presence of SCF. When the 104D2-FITC plus
104D2-Cy3-labeled MO7e cells were incubated in the presence of SCF at
4°C, energy transfer between the two fluorochromes was markedly
diminished, compared with results obtained at 37°C, suggesting that
the c-kit receptor does not dimerize as readily at 4°C as
at 37°C (Table 2).
To define the relationship between SCF concentration and c-kit
receptor dimerization, MO7e cells labeled with 104D2-FITC and with
104D2-Cy3 were exposed to a range of SCF concentrations, and
dimerization was assessed by FRET (Table
3). In two experiments, FRET was detected at a concentration of SCF
less than or equal to 10 ng/mL. Peak FRET was found at an SCF
concentration of 100 to 300 ng/mL. These results correlate well with
the concentrations of SCF that stimulate proliferation of MO7e cells
(Fig 1). Because the extent of FRET is a
function of the distance separating the donor-acceptor pairs of
fluorochromes and of the number of donor-acceptor pairs of
fluorochromes within a threshold distance of each other, these results
provide an index of c-kit receptor dimerization.
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| Fig 1.
Proliferation of MO7e cells in response to SCF
correlates with c-kit receptor dimerization detected by FRET.
MO7e cells were washed to remove GM-CSF and resuspended at a
concentration of 1 × 105 cells/mL in IMDM suppplemented
with 10% FCS and various concentrations of SCF (0 to 1,000 ng/mL;
 ), or 104D2 (0.5 µg/mL). After a 3-day incubation at 37°C, the
number of viable cells was counted. The data represent the mean (± standard error of mean [SEM]) of triplicate values from one
experiment. The wells supplemented with 104D2 contained 1.1 ± 0.2 × 105 MO7e cells/mL on day 3.
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The onset of c-kit receptor dimerization detectable by FRET was
assessed by incubating MO7e cells without or with SCF for varying
periods of time (Fig 2). In the absence of
SCF, no detectable change in energy transfer occurred between the
fluorochromes during the 60-minute period. However, c-kit
receptor dimerization was detectable within 3 minutes of SCF addition,
peaked at 30 minutes, and was still detectable at 60 minutes after SCF
addition (Fig 2).
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| Fig 2.
Dimerization of the c-kit receptor is detectable
within 3 minutes after the addition of SCF. MO7e cells were labeled
with 104D2-FITC plus 104D2-Cy3 for 45 minutes at 4°C, then
incubated in the absence () or presence ( ) of SCF (200 ng/mL) for
3 minutes, 10 minutes, 30 minutes, or 60 minutes at 37°C, fixed,
and analyzed as described in Table 2. Two additional experiments gave
similar results. Analysis of variance with a post hoc Student's
t-test was used to determine if the acceptor/donor ratios at
the various time points in the three experiments were different than
the values at time 0. In the presence of SCF, the acceptor/donor ratios
at 3 minutes, 10 minutes, 30 minutes, and 60 minutes were different than the acceptor/donor ratio at time 0 (P < .05).
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The specificity of cytokine-induced c-kit receptor dimerization
was assessed by incubating MO7e cells, UT-7/Epo cells, or UT-7/Tpo
cells with a number of cytokines (Tables 2 and
4). Although MO7e cells can proliferate in
response to SCF, GM-CSF, or IL-3, only SCF induced c-kit
receptor dimerization is detectable by FRET (Table 2).
MO7e cells do not respond to Epo, and Epo did not induce c-kit
receptor dimerization in this cell line (Table 2). Despite the
megakaryocytic features of the MO7e cell line and the ample display of
Epo receptors on normal megakaryocytes,40 subsequent
experiments showed that MO7e cells fail to bind 125I-Epo
(data not shown). The UT-7/Epo and the UT-7/Tpo cell lines, derived
from the parental UT-7 cell line by culture in Epo or in Tpo,
respectively,29,31 offer an opportunity to explore the
concept of receptor cross-talk in cells that are responsive to multiple
cytokines. SCF induced c-kit receptor dimerization detectable
by FRET in UT-7/Epo cells (Table 4). In each of five experiments with
UT-7/Epo cells, Epo appeared to modestly promote FRET between the
104D2-FITC and the 104D2-Cy3 antibodies, in comparison to the results
obtained with no added growth factor or GM-CSF (Table 4). In the
UT-7/Tpo cell line, only SCF was able to induce c-kit receptor
dimerization detectable by FRET. Although Tpo and GM-CSF can support
proliferation of UT-7/Tpo cells,31 neither of these
cytokines stimulated c-kit receptor dimerization (Table 4).
Binding of SCF induces c-kit receptor dimerization and
intermolecular tyrosine phosphorylation by the kinase domain of the receptor.15 Because of the suggestion that Epo could
stimulate dimerization of the c-kit receptor, the ability of
Epo to stimulate c-kit receptor tyrosine phosphorylation was
investigated (Fig 3). Exposure of UT-7/Epo
cells to SCF or to Epo for 10 minutes resulted in readily detectable
tyrosine phosphorylation of the c-kit receptor (Fig 3, upper
panel, lanes 2 and 3). The magnitude of c-kit receptor tyrosine
phosphorylation in the presence of SCF was greater than that found in
the presence of Epo. Exposure of UT-7/Epo cells to GM-CSF did not
induce c-kit receptor tyrosine phosphorylation (Fig 3, upper
panel, lane 4). In the UT-7/Tpo cell line, only SCF was found to
stimulate c-kit receptor tyrosine phosphorylation (Fig 3, upper
panel, lane 6). In accord with the results of the c-kit
receptor dimerization experiments, neither Tpo nor GM-CSF induced
c-kit receptor tyrosine phosphorylation in UT-7/Tpo cells (Fig
3, upper panel, lanes 7 and 8). Tyrosine phosphorylation of the
c-kit receptor in UT-7/Epo cells was detectable within 5 minutes of the addition of either SCF or Epo (Fig 3, lower panel).
These experiments demonstrating that Epo can induce tyrosine
phosphorylation of the c-kit receptor support the concept that
Epo can activate the c-kit receptor in the UT-7/Epo cell line.
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| Fig 3.
Upper panel: Epo induces tyrosine phosphorylation of the
c-kit receptor in UT-7/Epo cells. UT-7/Epo cells were exposed
to no added growth factor (lane 1), SCF (150 ng/mL, lane 2), Epo (2.5 U/mL, lane 3), or GM-CSF (5 ng/mL, lane 4) for 10 minutes at 37°C.
UT-7/Tpo cells were exposed to no added growth factor (lane 5), SCF
(lane 6), Tpo (25 ng/mL, lane 7), or GM-CSF (lane 8). Cell lysates were
immunoprecipitated with SR-1 anti-c-kit receptor monoclonal
antibody, subjected to SDS-PAGE, and blotted with the 4G10
antiphosphotyrosine monoclonal antibody. Molecular weight markers are
indicated. The arrow identifies the c-kit receptor. Two
additional experiments gave similar results. Lower panel: tyrosine
phosphorylation of the c-kit receptor in UT-7/Epo cells is
detectable within 5 minutes of Epo stimulation. UT-7/Epo cells were
exposed to no added growth factor (lane 1), SCF (150 ng/mL, lane 2) for
5 minutes, SCF (lane 3) for 10 minutes, Epo (2.5 U/mL, lane 4) for 5 minutes, Epo (lane 5) for 10 minutes, or GM-CSF (2.5 ng/mL) for 5 minutes at 37°C.
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After SCF binds, the SCF-c-kit receptor complex is
internalized.41,42 To assess the kinetics of c-kit
receptor reappearance on the cell surface, MO7e cells were exposed to
SCF for 1 hour, and c-kit receptor display on the cell surface
was examined by flow cytometry (Fig 4).
Exposure to SCF for 1 hour decreased c-kit receptor display on
the cell surface. When SCF was removed from the medium, c-kit
receptor display on the cell surface increased gradually over several
hours, but had not returned to its initial density by 4 hours after
removal of SCF (Fig 4). Blocking new protein synthesis by the addition
of cycloheximide prevented the increase in c-kit receptor
display on the cell surface (Fig 4), arguing that internalized
c-kit receptor does not recycle to the cell surface.
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| Fig 4.
Reappearance of the c-kit receptor on the cell
surface requires new protein synthesis. Display of the c-kit
receptor on MO7e cells proliferating in GM-CSF, before exposure to SCF,
is shown in the left portion of the figure. MO7e cells were incubated
with SCF (100 ng/mL) for 1 hour at 37°C to induce internalization
of the c-kit receptor. The cells were washed to remove SCF, and
reappearance of the c-kit receptor on the cell surface was
assessed by flow cytometry at various time points (0 to 4 hours) after
removal of SCF. MO7e cells were incubated in parallel in the presence of cycloheximide (10 µg/mL) to inhibit new protein synthesis. The
data are presented as the mean (± SEM) of triplicate values. The
cells incubated in the absence of cycloheximide displayed more cell
surface c-kit receptor than the cells incubated in the presence
of cycloheximide (*P < .01, Student's t-test).
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Confocal microscopy was used to determine whether exposure to SCF would
alter the localization of the c-kit receptor on the cell
surface (Fig 5). The images in Fig 5
represent optical slices of approximately 1 µm taken half-way through
the cell, to facilitate the distinction between surface and
intracellular c-kit receptor. Before exposure to SCF, the
c-kit receptor protein was widely distributed on the surface of
most of the MO7e cells (Fig 5A and B), and little cytoplasmic
c-kit receptor was detected (Fig 5B). By 3 minutes after
exposure to SCF, punctate fluorescent foci were detected in one region
of many of the cells, suggesting aggregation of c-kit receptors
at this site (Fig 5C and D). Thirty minutes after exposure to SCF,
little cell surface c-kit receptor was detected (Fig 5E and F),
and the majority of the c-kit receptor protein was found inside
the cell (Fig 5F).
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| Fig 5.
The c-kit receptor clusters on the cell surface
within 3 minutes of exposure to SCF. MO7e cells were exposed to SCF
(200 ng/mL) for 0 minutes (A and B), 3 minutes (C and D), or 30 minutes
(E and F) at 37°C. One portion of each aliquot of cells was stained with 104D2 followed by goat antimouse IgG-FITC to detect cell surface
c-kit receptor (A, C, and E). The other portion of each aliquot
of cells was permeabilized with Triton X-100 to permit detection of
intracellular c-kit receptor in addition to cell surface
c-kit receptor (B, D, and F), then stained as described above.
The cells were analyzed by confocal microscopy.
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To determine whether the FRET technique could detect c-kit
receptor dimerization in normal human hematopoietic cells, progeny of
peripheral blood BFU-E plucked on day 10 of culture were labeled with
104D2-FITC plus 104D2-Cy3 and incubated without or with SCF (Table 5). Although the mean fluorescence
intensity of 104D2 binding to the BFU-E progeny was substantially less
than the mean fluorescence intensity of 104D2 binding to the MO7e cell
line, SCF-induced dimerization of the c-kit receptor in the
BFU-E progeny was readily detectable (Table 5).
| |
DISCUSSION |
The c-kit receptor is a member of the tyrosine kinase type III
family of receptors, which includes the M-CSF receptor (c-fms), the platelet-derived growth factor (PDGF) receptors, and
the flt3/flk-2 receptor.15 These receptors are
characterized by an extracellular region that contains five
immunoglobulin-like domains, a single membrane-spanning region, and a
cytoplasmic region that encodes a tyrosine kinase domain split by a
kinase insert sequence. An essential early step in signal transduction
by this class of receptors is homodimerization and intermolecular
tyrosine phosphorylation. The present report shows that the FRET
technique offers one way to examine this proximal step in signal
transduction by the c-kit receptor.
Advantages of the FRET technique are that receptor homodimerization or
heterodimerization can be rapidly assessed in a population of living
cells.19-21,23,24 Small changes in energy transfer can be
accurately quantitated by flow cytometric analysis of thousands of
cells, and receptor dimerization can be investigated in the absence of
ligand. Thus, FRET can be used to study dynamic processes such as
assembly of receptor subunits within the cell membrane. Affinity
cross-linking, an alternative way to analyze receptor dimerization, is
usually performed in the presence of radiolabeled ligand to facilitate
identification of the cross-linked species.11,12 In at
least one situation, affinity cross-linking of 125I-Epo to
erythroid cells, the molecular weight of the major cross-linked proteins did not correspond to the molecular weight of the recombinant functional Epo receptor protein, and antibodies that recognize the
recombinant Epo receptor did not immunoprecipitate the major cross-linked proteins.43,44 The efficiency of affinity
cross-linking has been estimated to be in the range of 2% to
15%,22,45,46 and affinity cross-linking is less suitable
for kinetic analysis than is FRET.
Disadvantages of the FRET technique as used in this report include the
need for a nonneutralizing monoclonal antibody that recognizes the
receptor (or pair of antibodies in the case of receptor
heterodimerization) and that binds to an epitope that is accessible,
whether the receptor is a monomer or a dimer. An additional requirement
is that the orientation of the dimerized receptor must permit the
antibodies to be within the threshold distance of the fluorochrome pair
for FRET to occur. Although the lower limit of receptor display that
would permit detection of FRET has not been determined, the MO7e cell
line studied in this report displays approximately 35,000 c-kit
receptors per cell.47 A previous FRET study used cells
expressing approximately 10,000 IL-1 receptors per cell.21
However, the present report suggests that the level of c-kit
receptor display on normal hematopoietic precursor cells may be
sufficient for the application of the FRET technique to these
populations of cells.
The addition of SCF to human hematopoietic cell lines labeled with
104D2-FITC plus 104D2-Cy3 resulted in changes in mean fluorescence intensity that are in the same range as those detected in prior FRET
analysis of IL-1 receptor dimerization or of EGF receptor dimerization.21-23 Maximal IL-1 receptor dimerization
required at least 60 minutes after the addition of IL-121;
maximal c-kit receptor dimerization occurred more rapidly. When MO7e cells were maintained in the continued presence of SCF, the c-kit receptor remained dimerized for at least 1 hour (Fig 2). Flow cytometric analysis (Fig 4) and confocal microscopy (Fig 5) show
that a major portion of the c-kit receptor protein has been
internalized by 30 to 60 minutes after the addition of SCF. These
results suggest that at least a portion of the c-kit receptors remain dimerized after internalization. The EGF receptor also remains
dimerized after endocytosis,48 and it has been suggested that internalized tyrosine phosphorylated EGF receptors or PDGF receptors may participate in signal transduction.48,49
Normal endocytic trafficking of the EGF receptor is required to achieve the full spectrum of EGF signal transduction.50 Although
the present report suggests that internalized c-kit receptors
remain dimerized, whether internalized c-kit receptors
contribute to signal transduction remains to be determined.
Prior studies using FRET have shown that ligand binding leads to
microclustering of the EGF receptor in the cell membrane.23 Recent models of cell membrane structure suggest that certain signal
transduction proteins may be concentrated in lipid microdomains that
form "rafts" in the cell membrane, and that signaling may be
optimized by microclustering of receptors within these lipid microdomains.51 The T-cell receptor forms multimeric
complexes after engagement of antigenic peptide bound to major
histocompatibility complex (MHC) molecules, and the extent
of oligomerization of the T-cell receptor complex may influence signal
transduction.52 The confocal images in the present report
also suggest that the c-kit receptor aggregates in the presence
of SCF. Taken together, these reports suggest that the formation of
receptor aggregates is a common feature of a number of transmembrane
receptors.
Both SCF and Epo are important for normal erythropoiesis in vivo. Mice
that lack cell surface c-kit receptor (W/W mice) show a
profound reduction in fetal liver colony-forming unit-erythroid (CFU-E) numbers53 and die in the perinatal
period with severe anemia,54 indicating that SCF is
critical for normal late erythropoiesis in vivo. Likewise, Epo receptor
knock-out mice have BFU-E and CFU-E in the fetal liver, but die in
utero around embryonic day 13 with profound anemia.55,56
When the Epo receptor was introduced into fetal liver cells obtained
from Epo receptor knock-out mice, the cells required both SCF and Epo
for CFU-E growth in vitro, in contrast to normal fetal liver cells,
which require only Epo for CFU-E growth in vitro.26 These
results were interpreted to mean that an essential interaction between
the c-kit receptor and the Epo receptor normally occurs in
erythroid progenitor cells in vivo.26
Studies in the HCD57 murine hematopoietic cell line also suggest that
there is functional interaction between the c-kit receptor and
the Epo receptor.25,27 Stimulation of HCD57 cells with SCF
induced tyrosine phosphorylation of both the c-kit receptor and
the Epo receptor, and physical association of the c-kit
receptor with the Epo receptor cytoplasmic domain. However, these
reports do not indicate that Epo can trigger c-kit receptor
dimerization or tyrosine phosphorylation. The present report suggests
that Epo can induce c-kit receptor dimerization and shows that
Epo can induce c-kit receptor tyrosine phosphorylation in the
human hematopoietic cell line UT-7/Epo. This supports the concept of bidirectional cross-talk between the c-kit receptor and the Epo receptor.
Epo and Tpo exhibit a number of structural and functional similarities,
including approximately 50% amino acid homology between Epo and the
N-terminal domain of Tpo and conserved location of cysteine residues
and  helices.57 Epo and Tpo can synergistically promote
megakaryopoiesis (CFU-Meg colony growth) and erythropoiesis (CFU-E
generation) in vitro.58-60 Moreover, the Epo receptor and the Mpl receptor, both members of the hematopoietin receptor
superfamily, have amino acid sequence homology.61 Both the
Epo and the Mpl receptors are found on megakaryocytes and on erythroid
progenitor and precursor cells.34,40,62 Although both Epo
and Tpo can synergize with SCF, the mechanisms appear to differ in that
Epo, but not Tpo, was able to induce subtle changes in FRET suggestive of c-kit receptor dimerization; the latter finding corresponded to the ability of Epo, but not Tpo, to induce c-kit receptor
tyrosine phosphorylation in cytokine-responsive cell lines.
Confocal microscopy showed that the c-kit receptor can cap at
one end of the cell within 3 minutes after the addition of SCF, showing
that the c-kit receptor protein occupied by soluble SCF is
highly mobile within the cell membrane. Whether engagement of the
c-kit receptor by the transmembrane form of SCF presented by
marrow microenvironmental cells would permit equally rapid redistribution of the c-kit receptor on the hematopoietic cell surface remains to be determined. Capping may be related to
internalization via clathrin-coated pits; a number of other cytokine
receptors are internalized by the clathrin-coated pit
mechanism.63 After internalization, the c-kit
receptor protein is detectable within the cell by confocal microscopy
for at least 30 minutes. However, internalized c-kit receptor
does not redecorate the cell surface: reappearance of the c-kit
receptor on the cell surface requires new protein synthesis. These
results suggest that the fate of internalized c-kit receptor is
degradation, rather than recycling to the cell surface. It is likely
that both lysosomal proteolysis and the ubiquitin-proteasome pathway
contribute to c-kit receptor degradation.40,42,64
Receptor oligomerization is important for signal transduction in the
hematopoietin receptor superfamily,65 as well as in the
tyrosine kinase receptor family. With the use of distinct monoclonal
antibodies to the and  subunits of receptors, as has been done
for the T-cell antigen receptor complex,19,20 the FRET
technique could be used to examine heterodimerization of hematopoietic
growth factor receptors.
| |
FOOTNOTES |
Submitted June 5, 1997;
accepted October 3, 1997.
Supported by National Institutes of Health (Bethesda, MD) Grants No.
DK44194, DK49855, DK43719, ES07033, and a Faculty Research Award from
the American Cancer Society (Atlanta, GA).
Address reprint requests to Virginia C. Broudy, MD, Division of
Hematology, University of Washington, Box 357710, Seattle, WA
98195-7710.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Peter Rabinovitch for helpful discussions.
| |
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K. Li, C. Miller, S. Hegde, and D. Wojchowski
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U. Wojda, K. R. Leigh, J. M. Njoroge, K. A. Jackson, B. Natarajan, M. Stitely, and J. L. Miller
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C. D. Krause, E. Mei, J. Xie, Y. Jia, M. A. Bopp, R. M. Hochstrasser, and S. Pestka
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D. Locke, H. Chen, Y. Liu, C. Liu, and M. L. Kahn
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K. G. Otto, V. C. Broudy, N. L. Lin, E. Parganas, J. N. Luthi, J. G. Drachman, J. N. Ihle, and C. A. Blau
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T. B. van Dijk, E. van den Akker, M. P.-v. Amelsvoort, H. Mano, B. Lowenberg, and M. von Lindern
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V. C. Broudy, N. L. Lin, W. C. Liles, S. J. Corey, B. O'Laughlin, S. Mou, and D. Linnekin
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M. von Lindern, W. Zauner, G. Mellitzer, P. Steinlein, G. Fritsch, K. Huber, B. Lowenberg, and H. Beug
The Glucocorticoid Receptor Cooperates With the Erythropoietin Receptor and c-Kit to Enhance and Sustain Proliferation of Erythroid Progenitors In Vitro
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Abstract
Introduction
Abstract