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(The FASEB Journal. 2001;15:970-977.)
© 2001 FASEB

Fibroblast growth factor receptor 2 (FGFR2) in brain neurons and retinal pigment epithelial cells act via stimulation of neuroendocrine L-type channels (Cav1.3)

RITA ROSENTHAL, HAGEN THIEME and OLAF STRAUSS1

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
TOP
 ABSTRACT
INTRODUCTION
 METHODS AND MATERIALS
 REFERENCES
 
In contrast to the fibroblast growth factor receptor 1 (FGFR1), little is known about intracellular signaling of FGFR2. The signaling cascade of FGFR2 was studied using the perforated patch configuration of the patch-clamp technique in cultured rat retinal pigment epithelial (RPE) cells that express both FGFR1 and FGFR2. Interaction of signaling proteins was studied using immunoprecipitation techniques with membrane proteins from RPE cells and freshly isolated rat brain. When Ba2+ currents through L-type channels were studied, extracellular application of bFGF (10 ng/ml) led to a shift of the steady-state activation to more negative values. In 50% of cells, an additional increase in maximal current amplitude was observed. This effect was blocked by the tyrosine kinase inhibitor lavendustin A (10-5 M) but was not influenced by the FGFR1 blocker SU5402 (2x10-5 M) or by the blocker for src-kinase herbimycin A (10-5 M). Immunoprecipitation of FGFR2 led to coprecipitation of {alpha}1D Ca2+ channel subunits and precipitation of {alpha}1D subunits led to coprecipitation of FGFR2. Immunoprecipitation of FGFR1 did not result in the coprecipitation with {alpha}1D Ca2+ channel subunits. The coprecipitation results were comparable when using brain tissue and RPE cells. The {alpha}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 {alpha} 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
TOP
 ABSTRACT
 INTRODUCTION
METHODS AND MATERIALS
 REFERENCES
 
BASIC FIBROBLAST GROWTH factor (bFGF or FGF-2) has pleiotropic effects and plays a regulatory role in angiogenesis, smooth muscle cell growth or wound healing and promotes photoreceptor survival in the eye (1) . bFGF can act via two types of growth factor receptors: the FGFR1 (flg) and FGFR2 (bek). In contrast to signaling cascades involving FGFR1, intracellular signaling after stimulation of FGFR2 is poorly understood (2 , 3) . For FGFR1, intracellular signaling cascades involving phospholipase C, src family kinases, mitogen-activated kinases and the Grb2/Sos signaling complex have been described. Despite the close structural relation to the FGFR1, these intracellular signaling cascades have not been confirmed for FGFR2. In addition, it is unclear in which way Ca2+ channels are involved in bFGF-dependent intracellular signaling. It is known that bFGF can stimulate L-type Ca2+ channels and that stimulation of these channels is required for bFGF-induced changes in gene expression (4 5 6 7) .

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 {alpha}1D subunits of L-type Ca2+ channels in the RPE as well as in brain neurons.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
REFERENCES
 
Cell culture
Primary cultures of RPE cells from 6- to 8-day-old pigmented rats (BDE or hooded rats) were established as described previously according to the method of Edwards (19) . RPE cells were cultured in Ham’s F-10 culture medium supplemented with 20% fetal calf serum, 100 µg/ml kanamycin, and 50 µg/ml gentamycin. The cultures were maintained in 5% CO2 at 37°C. Primary cultures 6–10 days old were used for patch-clamp recordings and immunoprecipitation, since they express the highest density of L-type channels.

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 3–5 M{Omega} 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{Omega} (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 20–30 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 {alpha}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-{alpha}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-{alpha}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-{alpha}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 Student’s 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 3–10 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).



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  		Figure 1. Identification of Ba2+ currents through L-type Ca2+ channels. A) Pattern of electrical stimulation to induce inward currents. The membrane potential was clamped to -70 mV, the cell was stimulated by nine voltage steps of 50 ms duration and +10 mV increasing amplitudes. B) Inward currents induced by the electrical stimulation that appear in the presence of extracellular Ba2+ (10-2 M) under extra- and intracellular K+-free conditions. C) Effect of extracellular application of BayK 8644 (5x10-6 M) summarized in a current/voltage plot and as current traces (insert). For the current/voltage relation, maximal peak current amplitudes were plotted against the test potentials of the electrical stimulation.

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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  		Table 1. No caption available.



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  		Figure 2. Effect of extracellularly applied bFGF on L-type currents. A) Currents (induced by voltage step from -70 mV to +10 mV) measured directly after establishing the perforated patch configuration (0 min) and 4 min later (4 min) in the presence of bFGF (10 ng/ml). Cells were maintained serum-free for 24 h prior to patch-clamp recordings. B) Summary of experiments investigating the effect of bFGF. Maximal peak currents (voltage step from -70 mV to +10 mV) were measured every 2 min and plotted against the time. Currents were normalized to the value that was measured directly before application of bFGF. The upper trace was determined in the presence of bFGF (n=4) and the lower trace in the absence of bFGF (n=5). C) Effect of bFGF on steady-state activation and inactivation of L-type channels. Data were recorded at the maximum of the bFGF effect. Currents were normalized to the maximal current measured at a voltage step from -80 mV to +10 mV. Steady-state activation was determined using the electrical stimulation shown in Fig. 1A . To determine steady-state inactivation, maximal currents were measured at a voltage step to +10 mV from prepulse potentials that increased stepwise from -80 mV to +20 mV in 10 mV steps of 500 ms duration. Curves were fitted using the Boltzmann equation. bFGF (10 ng/ml; squares) led to a shift of the steady-state activation curve (circles) to a more negative voltage-range.

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).



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  		Figure 3. Effect of blockers for tyrosine kinase on the bFGF effect. A) Summary of the changes in the maximal peak current amplitude at the beginning of the perforated patch configuration and at the maximum of the bFGF effect: in the absence of bFGF, in the presence of bFGF (10 ng/ml), in the presence of bFGF and SU5402 (2 x 10-5 M; 5 min incubation prior bFGF application), in the presence of bFGF and lavendustin A (10-5 M; 5 min incubation prior to application of bFGF) and herbimycin A (10-5 M; incubation overnight prior application of bFGF). B) Effect of herbimycin A (10-5 M; incubation overnight prior to application of bFGF) on maximal L-type current amplitude. Maximal peak currents (voltage step from -70 mV to +10 mV) were measured every 2 min and plotted against the time of the experiment. Currents were normalized to the value measured directly before application of bFGF. The upper trace represents changes in the current amplitude in the presence of herbimycin A and bFGF, the lower trace only in the presence of herbimycin A. C) Effect of bFGF application in cells pretreated with herbimycin A (10-5 M; incubation overnight prior application of bFGF) on the steady-state activation and inactivation curves. Steady-state curves were determined as described in Fig. 2C . In the presence of herbimycin A, bFGF led mainly to a shift of the steady-state activation curve to a more negative voltage-range.

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 {alpha}1D subunits (~ 240 kDa) could also be stained using antiphosphotyrosine antibodies.



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  		Figure 4. Analysis of {alpha}1D subunit-specific membrane proteins from RPE cells and rat brain. A) Western blot analysis of membrane proteins. Left panels: Staining with anti-{alpha}1D subunit antibodies reveals the presence of {alpha} subunits from L-type channels of the neuroendocrine subtype in membrane proteins from both RPE cells (from serum-containing cultures) and rat brain tissue. Right panel: Staining of the same Western blot using antiphosphotyrosine antibodies (anti-p-tyr) shows the tyrosine phosphorylation of {alpha}1D subunit-specific bands. B) Western blot analysis of immunoprecipitates obtained using anti-{alpha}1D subunit antibodies (RPE cells from serum-containing cultures). Left panels: Staining with anti-{alpha}1D subunit antibodies shows the enrichment of proteins specific for {alpha} subunits of neuroendocrine L-type channels (arrows indicate the bands that disappear using the corresponding blocking peptide). Middle panels: Staining of the same precipitates using anti-FGFR2 antibodies reveals the coprecipitation of FGFR2 (arrows indicate the bands that disappear using the corresponding blocking peptide). Right panel: Staining with anti-FGFR1 antibodies did not result in the detection of FGFR1-specific proteins among these precipitates. C) Western blot analysis of immunoprecipitates obtained with anti-{alpha}1D subunit antibodies pretreated with the corresponding blocking peptide. Under these conditions no coprecipitation of FGFR2 with L-type channel {alpha} subunits occurred.

Immunoprecipitation
An {alpha}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 {alpha}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 {alpha}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 {alpha}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, {alpha}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 {alpha}1D subunit-specific proteins (Fig. 5C ). Similar results were obtained using membrane proteins from rat brain.



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  		Figure 5. Immunoprecipitation of bFGF receptors. A) Immunoprecipitation of membrane proteins from RPE cells and rat brain using antibodies against FGFR2 (bek). Left panels: Western blot analysis of precipitates stained with anti-FGFR2 antibodies shows the presence of FGFR2 (arrows indicate the bands that disappear using the corresponding blocking peptide). Right panels: Western blot analysis of the precipitates stained with anti-{alpha}1D subunit antibodies (arrows indicate the bands that disappear in the presence of the corresponding blocking peptide). The proteins were isolated from (left to right): rat brain, RPE cells maintained in serum-containing cultures, RPE cells maintained for 24 h in serum-free cultures prior immunoprecipitation experiments, RPE cells maintained in serum-free cultures incubated in bFGF (10 ng/ml; 1 h). Coprecipitation was not observed in precipitates obtained from RPE cells maintained in serum-free cultures. B) Immunoprecipitation with antibodies against FGFR2, where the antibodies were pretreated with the corresponding blocking peptide. Western blot analysis of the precipitates stained with anti-{alpha}1D subunit antibodies shows no coprecipitation with {alpha} subunits of L-type channels. C) Immunoprecipitation of membrane proteins from RPE cells and rat brain using antibodies against FGFR1 (flg). Left panels: Western blot analysis of the precipitates indicates the presence of FGFR1 (anti-FGFR1 staining was compared with the corresponding positive control). Right panels: Western blot analysis of the precipitates stained with anti-{alpha}1D subunit antibodies indicate no coprecipitation with {alpha} subunits from L-type channels.

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 {alpha} 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 {alpha}1D subunits, which correspond to the neuroendocrine subtype of L-type Ca2+ channels. Immunoprecipitation using anti-{alpha}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 {alpha}1D subunits is strengthened by immunoprecipitation experiments using anti-FGFR1 antibodies. In these experiments, no coprecipitation of FGFR1 and {alpha}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 {alpha}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 {alpha} 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 {alpha}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
 
This work was supported by DFG grant Str 480 3–1, Str 480/8-1 and Pro Retina. The technical assistance of M. Boxberger is gratefully acknowledged. The authors also thank Dr. F. Stumpff and Dr. O. Huber for helpful discussions.

Received for publication April 19, 2000. Revision received October 3, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 REFERENCES
 

  1. Bikfalvi, A., Klein, S., Pintucci, G., Rifkin, D. B. (1997) Biological roles of fibroblast growth factor-2. Endocr. Rev. 18,26-45[Abstract/Free Full Text]
  2. Friesel, R. E., Maciag, T. (1995) Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. FASEB J 9,919-925[Abstract]
  3. Klint, P., Claesson-Welsh, L. (1999) Signal transduction by fibroblast growth factor receptors. Frontiers Biosci 4,D165-D177[Medline]
  4. Zhu, D. L., Herembert, T., Caruelle, D., Caruelle, J. P., Marche, P. (1994) Involvement of calcium channels in fibroblast growth factor-induced activation of arterial cells in spontaneously hypertensive rats. J. Cardiovasc. Pharmacol. 23,395-400[Medline]
  5. Williams, E. J., Furness, J., Walsh, F. S., Doherty, P. (1994) Characterization of the second messenger pathway underlying neurite outgrowth stimulated by FGF. Development 120,1685-1693[Abstract]
  6. Puro, D. G., Mano, T. (1991) Modulation of calcium channels in human retinal glial cells by basic fibroblast growth factor: a possible role in retinal pathobiology. J. Neurosci. 11,1873-1880[Abstract]
  7. Koike, H., Saito, H., Matsuki, N. (1993) Effect of fibroblast growth factors on calcium currents in acutely isolated neuronal cells from rat ventromedial hypothalamus. Neurosci. Lett. 150,57-60[Medline]
  8. Tanihara, H., Inatani, M., Honda, Y. (1997) Growth factors and their receptors in the retina and pigment epithelium. Prog. Retinal Eye Res. 16,271-301
  9. Faktorovich, E. G., Steinberg, R. H., Yasumura, D., Matthes, M. T., LaVail, M. M. (1992) Basic fibroblast growth factor and local injury protect photoreceptors from light damage in the rat. J. Neurosci. 12,3554-3567[Abstract]
  10. LaVail, M. M., Unoki, K., Yasumura, D., Matthes, M. T., Yancopoulos, G. D., Steinberg, R. H. (1992) Multiple growth factors, cytokines and neurotrophins rescue photoreceptors from damaging effects of constant light. Proc. Natl. Acad. Sci. USA 89,11249-11253[Abstract/Free Full Text]
  11. Yoshida, M., Tanihara, H., Yoshimura, N. (1992) Platelet-derived growth factor gene expression in cultured human retinal pigment epithelial cells. Biochem. Biophys. Res. Commun. 189,66-71[Medline]
  12. Tanihara, H., Yoshida, M., Matsumoto, M., Yoshimura, N. (1993) Identification of transforming growth factor-beta expressed in cultured human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 34,413-419[Abstract/Free Full Text]
  13. Campochiaro, P. A., Hackett, S. F., Vinores, S. A., Freund, J., Csaky, C., LaRochelle, W., Henderer, J., Johnson, M., Rodriguez, I. R., Friedman, Z., et al (1994) Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells. J. Cell Sci. 107,2459-2469[Abstract]
  14. Liu, X., Ye, X., Yanoff, M., Li, W. (1998) Regulatory effects of soluble growth factors on choriocapillaris endothelial growth and survival. Ophthalmic Res 30,302-313[Medline]
  15. Bost, L. M., Aotaki-Keen, A. E., Hjelmeland, L. M. (1992) Coexpression of FGF-5 and bFGF by the retinal pigment epithelium in vitro. Exp. Eye Res. 55,727-734[Medline]
  16. Steele, F. R., Chader, G. J., Johnson, L. V., Tombran-Tink, J. (1992) Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc. Natl. Acad. Sci. USA 90,1526-1530[Abstract/Free Full Text]
  17. Guillonneau, X., Regnier-Ricard, F., Dupuis, C., Courtois, Y., Mascarelli, F. (1997) FGF2-stimulated release of endogenous FGF1 is associated with reduced apoptosis in retinal pigmented epithelial cells. Exp. Cell Res. 233,198-206[Medline]
  18. Mergler, S., Steinhausen, K., Wiederholt, M., Strauss, O. (1998) Altered regulation of L-type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration. FASEB J 12,1125-1134[Abstract/Free Full Text]
  19. Edwards, R. B. (1977) Culture of rat retinal pigment epithelium. In Vitro 13,301-304[Medline]
  20. Ueda, Y., Steinberg, R. H. (1993) Voltage-operated calcium channels in fresh and cultured rat retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 34,3408-3418[Abstract/Free Full Text]
  21. Ueda, Y., Steinberg, R. H. (1995) Dihydropyridine-sensitive calcium currents in freshly isolated human and monkey retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 36,373-380[Abstract/Free Full Text]
  22. Strauss, O., Wienrich, M. (1994) Ca2+ conductances in cultured rat retinal pigment epithelial cells. J. Cell. Physiol. 160,89-96[Medline]
  23. Strauss, O., Weiser, T., Wienrich, M. (1994) Potassium currents in cultured cells of the rat retinal pigment epithelium. Comp. Biochem. Physiol. A. Physiol. 109A,975-983[Medline]
  24. Wen, R., Lui, G. M., Steinberg, R. H. (1993) Whole-cell K+ currents in fresh and cultured cells of the human and the monkey retinal pigment epithelium. J. Physiol. 465,121-147[Abstract/Free Full Text]
  25. Strauss, O., Richard, G., Wienrich, M. (1993) Voltage-dependent potassium currents in cultured human retinal pigment epithelial cells. Biochem. Biophys. Res. Commun. 191,775-781[Medline]
  26. Hughes, B. A., Steinberg, R. H. (1990) Voltage-dependent currents in isolated cells of the frog retinal pigment epithelium. J. Physiol. 428,273-297[Abstract/Free Full Text]
  27. Tao, Q., Rafuse, P. E., Kelly, M. E. M. (1994) Potassium currents in cultured rabbit retinal pigment epithelial cells. J. Membr. Biol. 141,123-138[Medline]
  28. Kuriyama, S., Ohuchi, T., Yoshimura, N., Honda, Y. (1991) Growth factor-induced cytosolic calcium ion transients in cultured human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 32,2882-2890[Abstract/Free Full Text]
  29. Strauss, O., Mergler, S., Wiederholt, M. (1997) Regulation of L-type calcium channels by tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells. FASEB J 11,859-867[Abstract]
  30. Mohammadi, M., McMahon, G., Sun, L., Tang, C., Hirth, P., Yeh, B. K., Hubbard, S. R., Schlessinger, J. (1997) Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276,955-960[Abstract/Free Full Text]
  31. Ahlijanian, M. K., Westenbroek, R. E., Catterall, W. A. (1990) Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord, and retina. Neuron 4,819-832[Medline]
  32. Westenbroek, R. E., Ahlijanian, M. K., Catterall, W. A. (1990) Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons. Nature (London) 347,281-284[Medline]
  33. Bito, H., Deisseroth, K., Tsien, R. W. (1996) CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87,1203-1214[Medline]
  34. Catterall, W. A. (1998) Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium 24,307-323[Medline]
  35. Deisseroth, K., Heist, E. K., Tsien, R. W. (1998) Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature (London) 392,198-202[Medline]
  36. Murphy, T. H., Worley, P. F., Baraban, J. M. (1991) L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes. Neuron 7,625-635[Medline]
  37. Rosen, L. B., Ginty, D. D., Greenberg, M. E. (1995) Calcium regulation of gene expression. Adv. Second Messenger Phosphoprotein Res. 30,225-253[Medline]
  38. Ishigooka, H., Kitaoka, T., Boutilier, S. B., Bost, L. M., Aotaki-Keen, A. E., Tablin, F., Hjelmeland, L. M. (1993) Developmental expression of bFGF in the bovine retina. Invest. Ophthalmol. Vis. Sci. 34,2813-2823[Abstract/Free Full Text]



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