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Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 12200 Berlin, Germany
1Correspondence: Institut f. Klinische Physiologie, Universitaetsklinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: strauss{at}ukbf.fu-berlin.de
| ABSTRACT |
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1D Ca2+ channel subunits and
precipitation of
1D subunits led to coprecipitation of FGFR2.
Immunoprecipitation of FGFR1 did not result in the coprecipitation with
1D Ca2+ channel subunits. The coprecipitation results
were comparable when using brain tissue and RPE cells. The
1D
subunit-specific band were stained with antiphosphotyrosine antibodies.
We conclude that FGFR2 acts via a different signaling cascade than
FGFR1. This cascade involves an src-kinase-independent, close
functional interaction of FGFR2 and the
subunit of neuroendocrine
L-type channels.Rosenthal, R., Thieme, H., Strauss, O. Fibroblast
growth factor receptor 2 (FGFR2) in brain neurons and retinal pigment
epithelial cells act via stimulation of neuroendocrine L-type channels
(Cav1.3).
Key Words: RPE bek flg Ca2+ channels bFGF
| INTRODUCTION |
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Among other functions, the retinal pigment epithelium (RPE) secretes a
variety of growth factors that help to maintain the structural
integrity of photoreceptors (8
9
10
11
12
13
14
15
16)
. This secretion is
auto- and paracrinially controlled by growth factors (8)
.
For example, bFGF stimulates the secretion of acidic fibroblast growth
factor (aFGF or FGF-1) (17)
. In these cells, we were able
to show that bFGF induces a rise in cytosolic free calcium due to a
tyrosine kinase-dependent activation of L-type channels
(18)
. Furthermore, a changed tyrosine kinase-dependent
stimulation of L-type channels seemed to be involved in the etiology of
an inherited retinal degeneration that is accompanied by a changed bFGF
secretion (18)
. The purpose of this study is to
characterize bFGF-dependent intracellular signaling in the RPE that
express both FGFR1 and FGFR2. We found that FGFR2-dependent
intracellular signaling is different from FGFR1 signaling and involves
direct stimulation of
1D subunits of L-type
Ca2+ channels in the RPE as well as in brain
neurons.
| METHODS AND MATERIALS |
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Perforated patch-clamp recordings
Electrophysiological recordings were performed at room
temperature. Coverslips with RPE cells were mounted on the stage of an
inverted microscope and superfused with a bath solution containing (in
10-3 M) 130 NaCl, 3 tetraethylammonium chloride,
0.3 CaCl2, 0.6 MgCl2, 14
NaHCO3, 1
Na2HPO4, 33 HEPES, and 6
glucose (pH=7.2 with Tris). The bath solution contained
10-2 M BaCl2 as charge
carrier to measure currents through L-type channels. All drugs were
added from DMSO containing stock solutions. The DMSO concentration in
the bath solution was lower than 0.1%. This concentration had no
influence on the amplitude of L-type currents in RPE cells (max.
amplitude of 1±0.2 pApF-1; n=3).
Pipettes of borosilicate glass with a resistance of 35 M
were
pulled using a Universal Puller (Zeitz, Augsburg, Germany). Pipettes
were filled with pipette solution containing (in
10-3 M) 100 CsCl, 10 NaCl, 0.5
CaCl2, 2 MgCl2, 5.5
EGTA-Tris, 10 HEPES (pH=7.2 with Tris). For perforated patch
recordings, the pipette solution contained 150 µg/ml nystatin. The
osmolality of the pipette solution was 280 ± 4 mOsmol
(SE; n=3) and of the bath solution
312 ± 6 mOsmol (SE; n=3;
estimated using the Vapor Pressure Osmometer 5100B, Wescor, Logan,
Utah). Membrane currents were recorded using an EPC-7 patch-clamp
amplifier (HEKA, Lamprecht, Germany). Electrical stimulation, data
storage, and data processing were performed using TIDA (HEKA,
Lamprecht, Germany) software in conjunction with an AT-compatible
computer. Membrane capacitance and access resistance were calculated
from the capacitance current transient induced by a depolarizing
voltage step of 30 ms duration and 10 mV amplitude from the holding
potential. In the perforated patch configuration, the cells showed an
access resistance of 26.7 ± 1.8 M
(SE,
n=25). The mean membrane capacitance in RPE cells was
155 ± 4.7 pF (SE, n=20). Access
resistance and membrane capacitance were compensated by the patch-clamp
amplifier. Potentials were automatically corrected for liquid junction
potential (+1.5 mV) by the patch-clamp software. Voltage-dependent
barium currents were induced by 9 depolarizing voltage steps of 50 ms
and 10 mV increasing amplitude at a frequency of 1 Hz from a holding
potential of -70 mV. Steady-state activation and inactivation curves
were fitted using the Boltzmann equation.
Preparation of membrane proteins and immunoprecipitation
Cell lysis (RPE cells: cultures from 2030 eyes; brain: 100 mg
fresh tissue) was performed in lysis buffer containing
(10-3 M) 150 NaCl, 50 Tris, 1 Na-orthovanadate,
0.05 NaF (pH=7.6 with HCl). The lysis buffer was supplemented with 1%
Triton X-100, 0.5% Na-desoxycholate, 0.1 SDS, and 0.3 µg/ml EDTA.
For protease inhibition, lysis buffer contained (µg/ml) 16
benzamidine-HCl, 10 phenanthroline, 10 leupeptin, 10 pepstatin, 174
phenylmethylsulfonylfluoride, 1 aprotinin. Cell lysis was performed
using a Polytron homogenizer (Kinematik, Switzerland) and by three
freezing and thawing steps (liquid N2; 42°C).
Cell lysates were centrifuged at 5000 rpm (2380 g) for 5 min
at 4°C. The supernatant was centrifuged a second time at 18,000 rpm
(40,000 g) for 30 min. The pellet was suspended in lysis
buffer incubated overnight at 4°C with Sepharose beads carrying
antibodies against
1D Ca2+ channel subunits,
FGFR1 (flg) or FGFR2 (bek) (prewashed Sepharose beads incubated with
antibodies for 1 h at 4°C). For control experiments beads were
incubated with antibodies together with the corresponding blocking
peptides (anti-
1D subunits: blocking peptide: 1:1; anti-FGFR2 :
blocking peptide: 1:5). Beads were washed and sampled by centrifugation
and suspended in 1 x Laemmli buffer. To separate precipitates
from Sepharose beads, the suspension was incubated at 95°C for 5 min
and centrifuged at 14,000 rpm (18,620 g) for 2 min. The
supernatant was subjected to Western blot analysis.
Western blot analysis
Membrane proteins or immunoprecipitates were separated by
polyacrylamide gel electrophoresis (8.5% polyacrylamide). Equal
amounts of protein were loaded in each lane of the gels (
30 µg of
total protein). Electrophoresis was performed in Mini-Protean cells
(Bio-Rad Life Science Group, Hercules, Calif.) for 1 h at 150 V.
The proteins were then blotted to nitrocellulose filter screens
(Polyscreen, NENTM, Life Science Products Boston, Mass.) for 1 h
at 100 V. Protein blots were blocked in PBS/Tween supplemented with
non-fat dry milk (10%) for 2 h and with BSA (5%) for 4 h at
room temperature. The blots were probed overnight at 4°C with
antibodies against Ca2+ channel subunits, FGF
receptors or phosphotyrosine residues and incubated with
peroxidase-conjugated secondary antibody for 1 h at room
temperature. The blots were visualized using a chemiluminescence kit
(Amersham Pharmacia Biotech, Braunschweig, Germany). Blots were
digitalized using the LAS-1000 Image Analyzer (Fujifilm, Berlin,
Germany) and the AIDA 2.0 software (Raytest, Berlin, Germany) in
conjunction with an AT-compatible computer. To verify specific staining
of protein bands Western blots were stripped and stained a second time
using the same antibody together with the corresponding blocking
peptide (anti-
1D subunits: blocking peptide, 1:1; anti-FGFR2:
blocking peptide: 1:5).
Chemicals
Media and cell culture supplements were purchased from Gibco Life
Technologies, Inc. (Eggenstein, Germany). All chemicals were purchased
from Sigma (Munich, Germany), Research Biochemicals (Köln,
Germany), Serva (Heidelberg, Germany), and Merck (Darmstadt, Germany).
The following antibodies were obtained: anti-
1D from Alomone
Laboratories (München, Germany), anti-FGFR2 (anti-bek C17) from
Santa Cruz (Heidelberg, Germany), peroxidase-conjugated secondary
antibody from Dianova (Hamburg, Germany), and anti-FGFR1 from Biomol
(Hamburg, Germany).
Statistical analysis
Data were presented as mean ± SE and analyzed
for significance by the unpaired Students t test. Mean
values of data from Boltzmann fits were calculated from individual fits
of each experiment. Data were considered to be significantly different
at P values below 0.05. All protein biochemical experiments
were performed at least three or four times and all
electrophysiological recordings were performed 310 times; unless
otherwise stated the figures show one representative experiment.
RESULTS
Measurement of L-type currents
Under extra- and intracellular K+-free
conditions, depolarization of RPE cells led to
Ba2+ inward currents that display characteristics
of L-type Ca2+ channels (Fig. 1
). They activate at potentials more positive than -30 mV to -20 mV,
display a slow inactivation (t=110±10 ms; n=12), and can be
increased to 143.4 ± 7.5% of control (recovery to 105.4±8.6%;
n=6) using the dihydropyridine compound BayK 8644
(5x10-6 M).
|
For the following experiments, cells were maintained under serum-free
conditions for 24 h before the experiments. When bFGF (10 ng/ml)
was administered, a change in the voltage dependence of the L-type
channel currents could be observed in all cells, shifting
V1/2 of the steady-state activation curve from
-12 mV to the more negative value of -19 mV (Table 1
). bFGF led to no significant changes in steady-state inactivation
(Fig. 2
); 50% of investigated cells responded to bFGF application with an
additional increase in the maximal current amplitude (Fig. 2
; Table 1
)
of the value observed before application of bFGF. This maximal effect
of bFGF was observed after 4 min. Without bFGF, a slight decrease in
the maximal current amplitude was observed after establishing the
perforated patch configuration.
|
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The effect could be blocked entirely by adding lavendustin A
(10-5 M), a broad-range tyrosine kinase
inhibitor (Table 1
, Fig. 3
). In contrast, incubation of the cells in herbimycin A (blocker of the
cytosolic src subtype tyrosine kinase; 10-5 M)
for 12 h prior to patch-clamp experiments or application of SU5402
(blocker of FGFR1; 2x10-5 M) just before
bFGF application had no effect on the bFGF-induced rise in L-type
currents and shift of the steady-state activation curve (Table 1
, Fig. 3
). In the presence of SU 5402 or herbimycin A, 50% of investigated
cells responded to bFGF application with the additional increase in the
maximal current amplitude. The bFGF-induced increase in the maximal
current amplitude in the presence of SU5402 is not significantly
different from the one without SU5402 (P>0.05). We did,
however, observe a reduction in the L-type current density (peak
current induced by voltage step from -70 mV to +10 mV) after
incubation of the cell in herbimycin A from 1.4 ± 0.1
pApF-1 (n=20) to 0.9 ± 0.1
pApF-1 (n=16; P<0.01).
|
Western blot
Western blot techniques were used to identify
tyrosine-phosphorylated membrane proteins (Fig. 4A
). In membrane proteins from rat brain and RPE cells kept in
serum-containing cultures, the proteins were stained using antibodies
against
1D subunits (
240 kDa) could also be stained using
antiphosphotyrosine antibodies.
|
Immunoprecipitation
An
1D subunit-specific signal of
240 kDa could be
identified in immunoprecipitates obtained from membrane proteins of RPE
cells (kept in serum-containing culture medium) using antibodies
against
1D subunits of Ca2+ channel proteins
(Fig. 4B
, C
). In these immunoprecipitates, FGFR2-specific
proteins could be identified. In Western blots of membrane proteins
from RPE cells (kept in serum-containing medium) precipitated using
antibodies against FGFR2, not only could FGFR2-specific proteins be
identified (
120 kDa), but
1D subunit-specific proteins were also
detectable (
240 kDa; Fig. 5
). This observation could not been made when RPE cells were maintained
in serum-free medium for 24 h before immunoprecipitation. In
precipitates obtained from these cells using antibodies against FGFR2,
no coprecipitation with
1D subunits could be detected. In the next
set of experiments, cells kept in serum-free culture medium for 24 h were stimulated with bFGF (10 ng/ml; for 1 h). In these cells,
1D subunit-specific proteins were identified in membrane proteins
precipitated with antibodies against FGFR2. The specific staining in
Western blots was confirmed using blocking peptides. To demonstrate
that coprecipitation was not due to unspecific binding by antibodies,
control experiments were performed in which antibodies were treated
with blocking peptide before immunoprecipitation; here, no
coprecipitation could be detected. Immunoprecipitation of FGFR1 from
membrane proteins of RPE cells led to enrichment of FGFR1-specific
proteins (110 kDa) but not to coprecipitation of
1D subunit-specific
proteins (Fig. 5C
). Similar results were obtained using
membrane proteins from rat brain.
|
DISCUSSION
In this study we found that stimulation of FGFR2 leads to an
activation of L-type channels of the neuroendocrine subtype. This
activation involves a close interaction of FGFR2 with
subunits of
these Ca2+ channels of both rat RPE and brain
tissue. These findings are in marked contrast to the signaling cascades
that reportedly follow the activation of FGFR1 (2
, 3)
.
In serum-deprived RPE cells, extracellular application of bFGF led to
changes in voltage-dependent Ba2+ inward
currents. Since these Ba2+ currents activate at
potentials more positive than -30 mV, display a slow inactivation, and
are increased by the dihydropyridine compound BayK 8644, they could be
identified as currents through L-type Ca2+
channels. L-type channels have been described as
Ca2+ channels characteristic of RPE cells
(20
21
22)
. bFGF mainly led to a shift of the steady-state
activation curve to more negative potentials, closer to the resting
potential of RPE cells (
-45 mV) (23
24
25
26
27)
. In 50% of
the cells investigated, bFGF led to an additional increase in the
maximal current amplitude. This effect is comparable to bFGF-dependent
stimulation of L-type channels in other tissues such as neurons or glia
(6
, 7)
. The observation that the response to stimulation
by bFGF varies from cell to cell agrees well with other groups that
investigated cytosolic Ca2+ transients in RPE
cells or Ca2+ currents in neurons (7
, 28)
. These groups discussed the existence of subpopulations of
cells with different functional capacities. The reason for this is
unclear.
Application of the tyrosine kinase blocker lavendustin A prevented the
effect of bFGF, which indicates that the effect of bFGF is tyrosine
kinase dependent. This fits well with previously published observations
in which L-type channels appeared to be activated by the tyrosine
kinase pp60c-src (29)
. However,
preincubation of cells with the src-kinase inhibitor herbimycin A
overnight prior to patch-clamp experiments led to the expected
reduction in the current density of L-type channels, but could not
prevent the bFGF-induced shift of the steady-state activation curve or
increase in maximal current amplitude. Thus, src-kinase seems not to be
involved in the bFGF-induced activation of L-type channels. Since it is
known that RPE cells express both FGFR1 and FGFR2 (8)
, the
effect of bFGF can occur via one or both receptors. SU5402, a FGFR1
blocker (30)
, did not interfere with the response to bFGF.
Thus, FGFR1 does not seem to be involved in the effect of bFGF. The
involvement of FGFR2 could be demonstrated in immunoprecipitation
experiments with membrane proteins isolated from cultured RPE cells
from serum-containing cell culture or freshly isolated rat brain.
Immunoprecipitation using anti-FGFR2 antibodies led to coprecipitation
of
1D subunits, which correspond to the neuroendocrine subtype of
L-type Ca2+ channels. Immunoprecipitation using
anti-
1D subunit antibodies led to coprecipitation of FGFR2. Control
experiments in which the antibodies were pretreated with the
corresponding blocking peptides showed that the coprecipitation was not
due to unspecific binding by the antibodies. Here, no coprecipitation
could be observed.
That immunoprecipitation data reflect a functional interaction between
FGFR2 and
1D subunits is strengthened by immunoprecipitation
experiments using anti-FGFR1 antibodies. In these experiments, no
coprecipitation of FGFR1 and
1D subunits could be observed matching
the observations made in patch-clamp experiments using the FGFR1
blocker SU5402 (30)
, where no influence on the effect of
bFGF was observed. No coprecipitation of FGFR2 and
1D subunits was
observed in membrane proteins from cells kept in serum-free culture
medium for 24 h. The coprecipiation could be observed again when
cells from serum-free cultures were stimulated with bFGF. Thus, bFGF
acts via a close functional interaction of FGFR2 and
subunits of
neuroendocrine L-type channels.
In contrast to intracellular signaling induced by stimulation of FGFR1,
the intracellular signaling of FGFR2 is poorly understood (2
, 3)
. In this study, we could show that FGFR2 acts via a different
signaling cascade than FGFR1 even though both receptors are closely
related. This cascade involves the activation of the neuroendocrine
subtype of L-type channels by a tyrosine kinase that does not belong to
the src subtype. In addition, this cascade involves a close interaction
of FGFR2 and the
1 subunit of the Ca2+
channel. Ca2+ channels of the neuroendocrine
subtype are equally distributed over the dendrites and the cell body of
neurons (31
, 32)
, and seem to be mainly involved in the
activity-dependent regulation of gene expression via phosphorylation of
CREB (cyclic AMP-responsive element binding protein)
(33
34
35
36
37)
. It is noteworthy that the elevation of cytosolic
calcium levels by bFGF via activation of L-type channels has already
been demonstrated in RPE cells (18)
, neurons
(7)
, or glia cells (6)
. Thus, mechanisms for
the control of gene expression include not only activation of L-type
channels by neuronal activity, but also by stimulation of FGFR2. In RPE
cells that express both FGFR1 and FGFR2, bFGF has been shown to lead to
different changes in cell function such as changes in growth factor
secretion and cell differentiation (17
, 38)
. These
different cell functions may be regulated by the different signal
transduction cascades induced by FGFR1 and FGFR2.
| ACKNOWLEDGMENTS |
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Received for publication April 19, 2000.
Revision received October 3, 2000.
| REFERENCES |
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