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Originally published In Press as doi:10.1074/jbc.M311687200 on November 20, 2003

J. Biol. Chem., Vol. 279, Issue 7, 5852-5860, February 13, 2004
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Sensitization of Epidermal Growth Factor-induced Signaling by Bradykinin Is Mediated by c-Src

IMPLICATIONS FOR A ROLE OF LIPID MICRODOMAINS*

Eun-Mi Hur, Yong-Soo Park, Byoung Dae Lee, Il Ho Jang, Hyeon Soo Kim, Tae-Don Kim, Pann-Ghill Suh, Sung Ho Ryu, and Kyong-Tai Kim{ddagger}

From the Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang 790-784, Republic of Korea

Received for publication, October 24, 2003 , and in revised form, November 19, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Communication between receptor tyrosine kinase (RTK)- and G protein-coupled receptor (GPCR)-mediated signaling systems has received increasing attention in recent years. Here, we report that activation of G protein-coupled bradykinin B2 receptor induces an up-regulation of cellular responses mediated by epidermal growth factor receptor (EGFR) and provide essential mechanistic characteristics of this sensitization process. EGF, which failed to evoke detectable amount of calcium increase and neurotransmitter release when administrated alone in primary cultures of rat adrenal chromaffin cells and PC12 cells, became capable of inducing these responses specifically after bradykinin pretreatment. Both EGFR and non-receptor tyrosine kinase p60Src, whose kinase activities were required in the sensitization, were found to be enriched in cholesterol-rich lipid rafts. Bradykinin caused activation of p60Src and Src-dependent phosphorylation of the EGFR on Tyr-845 in lipid rafts, as well as recruitment of phospholipase C (PLC) {gamma}1 to the rafts. Depletion of cholesterol by methyl-{beta}-cyclodextrin disrupted the raft localization of EGFR and Src, as well as bradykinin-induced translocation of PLC{gamma}1. Furthermore, sensitization, which was impaired by cholesterol depletion, was restored by repletion of cholesterol. Therefore, we suggest that lipid rafts are essential participants in the regulation of receptor-mediated signal transduction and cross-talk via organizing signaling complexes in membrane microdomains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium plays an important role in regulating a great variety of neuronal processes, including excitability, associativity, gene transcription, synaptic plasticity, and neurotransmitter release (1). Differences in spatial, temporal, as well as quantitative aspects of calcium signals allow selective targeting of the Ca2+ signal from specific stimuli to appropriate responses. Growing evidence suggests that specificity and sensitivity of the Ca2+ signal is determined by organizing signaling molecules into microdomains in which Ca2+ signaling functions are carried out, providing a new concept in neuronal calcium signaling (2-4). Localized environments are, therefore, richly endowed with the versatility needed to allow Ca2+ increase to effectively couple specific receptors to effector molecules (5).

The plasma membrane contains lipid rafts, which are structurally and biochemically isolated cellular compartments implicated in the organization of specialized proteins into a series of discrete microdomains (6). A large number of receptor and non-receptor tyrosine kinases, G protein-coupled receptors (GPCRs),1 G proteins, and effector enzymes have been reported to present in or recruit to lipid rafts following activation. The functional significance of this localization has not been unequivocally demonstrated, but it seems to facilitate the association of appropriate signaling proteins (7, 8). In neuronal cells, compelling evidence is emerging implicating that lipid rafts act as platforms for localized signal transduction and that rafts are important for neural development and function, which include axon guidance and vesicular trafficking (9).

Epidermal growth factor (EGF) itself has been found unable to induce detectable amount of increase in intracellular calcium concentration ([Ca2+]i) in various cell types (10, 11). Interestingly, EGF becomes capable of inducing a substantial amount of increase in [Ca2+]i after bradykinin (BK) pretreatment in Swiss 3T3 mouse fibroblasts (10), SK-N-BE human neuroblastoma cells, and PC12 rat pheochromocytoma cells (11). However, to date, the mechanism underlying this "sensitization" of the EGF-induced [Ca2+]i increase has remained undetermined. In the present study, we report that EGF-induced neurotransmitter release as well as calcium mobilization is greatly sensitized specifically by BK in neuroendocrine cells. We postulate that the assemblage of signaling components into discrete microdomains may permit specific receptor cross-talk and render precise modulation of the downstream signaling events. To test this hypothesis, we investigated the possible role of lipid rafts in the communication between the G protein-coupled BK receptor- and the epidermal growth factor receptor (EGFR)-mediated signaling pathways. To our knowledge, this study is the first demonstration of providing evidence that BK induces Src-dependent phosphorylation of the EGFR as well as activation of p60Src in lipid rafts, and recruitment of phospholipase C (PLC) {gamma}1 to the rafts. We suggest that BK-induced sensitization of the EGF pathway requires the integrity of lipid raft structures, thereby providing a novel insight into how signal transduction pathways that employ distinct receptors and effectors influence each other.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fura-2 pentaacetoxymethyl ester (Fura-2/AM) was from Molecular Probes (Eugene, OR). Phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and protein G-agarose suspension were purchased from Roche Molecular Biochemicals (Mannheim, Germany). DArg[Hyp3,Thi5,DTic7,Oic8]bradykinin (HOE140), and SK&F96365 were from Research Biochemical International (Natick, MA). EGF was from Daewoong Pharmaceutical Co. (Seoul, South Korea). U73122 [GenBank] and U73343 [GenBank] were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). [3H]Inositol 1,4,5-trisphosphate was obtained from PerkinElmer Life Sciences (Boston, MA). The enhanced chemiluminescence (ECL) kit was from Amersham Biosciences (Buckinghamshire, UK). Anti-phosphotyrosine mAb (4G10) and anti-EGFR polyclonal antibody were from Upstate Biotechnology (Lake Placid, NY). Anti-BiP/GRP78 mAb and anti-flotillin-1 mAb were from BD Transduction Laboratories (Lexington, KY). Anti-caveolin-1 polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Src pTyr-418 and anti-EGFR pTyr-845 were from BioSource International, Inc. (Camarillo, CA). Anti-PLC{gamma}1 mAb was generated as previously described (12, 13). Anti-Src antibody, AG1478, ionomycin, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) were from Calbiochem (La Jolla, CA). Peroxidase-conjugated anti-mouse immunoglobulin G (IgG) and anti-sheep IgG were from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MA). All other reagents were from Sigma (St. Louis, MO).

Isolation and Culture of Rat Adrenal Medulla Chromaffin Cells—Rat chromaffin medullary cells were prepared as previously described (14, 15) with minor modifications. Briefly, ether-anesthetized female rats (200-250 g) were decapitated, and their adrenal glands were removed, dissected free of the cortex, and rinsed in HEPES buffer (137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM HEPES, 10 mM glucose, pH 7.4). Cells were dissociated by drawing adrenal tissue fragments gently up and down with a Pasteur pipette after treating them at 37 °C with 0.5 unit/ml collagenase I (Worthington Biochemical Corp., Lakewood, NJ) and 20 µg/ml DNase I (Sigma) for 30 min. After centrifugation and rinsing with the HEPES buffer, cells were suspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, plated on poly-D-lysine-coated glass coverslips, and cultured for 1-2 days under a 5% CO2-containing atmosphere.

Cell Culture—Rat pheochromocytoma PC12 cells were grown in RPMI 1640 medium as described previously (16). Cells at 70-80% confluence were growth-arrested by incubation in serum-free RPMI 1640 medium overnight prior to use. All experiments were performed with growth-arrested cells.

Plasmids and Transfection—The cDNA encoding a kinase-defective, dominant inhibitory form of Src (SrcK298M) cloned into mammalian expression vector pME-18S (17) was a generous gift from Drs. Tadashi Yamamoto (University of Tokyo, Japan) and Hiroshi Nishina (University of Tokyo, Japan). The pcDNA3 expression vector for the mutant EGFR Y845F (18, 19) was a generous gift from Dr. Sarah J. Parsons (University of Virginia). PC12 cells were transiently transfected with expression vectors by electroporation. In some experiments, cells were co-transfected with an expression vector for green fluorescent protein (Clontech, Franklin Lakes, NJ), and positive clones were identified by fluorescence microscopy to compare the transfection efficiencies between experiments.

Measurement of [Ca2+]i and Its Imaging—The level of [Ca2+]i was measured as described previously (16). Calcium imaging experiments were performed with a monochromator-based spectrophotofluorimetric system (DeltaScan Illumination System, PTI, South Brunswick, NJ). Cells grown on poly-D-lysine-coated glass coverslips were loaded with 4 µM fura-2/AM in serum-free growth medium (37 °C, 30 min). Coverslips then were placed on the stage of a Nikon Eclipse TE300 microscope, and intracellular Fura-2/AM was excited at 340 and 380 nm with a xenon lamp (USHIO, Tokyo, Japan). Intensified charge-coupled device video camera (IC-200, PTI) was used to collect information. The fluorescence signal at 510 nm was collected, and the ratio of the fluorescence at the two excitation wavelengths was calculated by ImagemasterTM software (version 1.4, PTI). Areas on the same coverslips without cells were recorded as background images.

Measurement of Inositol 1,4,5-Trisphosphate—Concentration of IP3 in cells was determined by competition assay with [3H]IP3 as we previously described (20).

Amperometric Measurement—Dopamine was loaded into PC12 cells, and electrophysiological recording conditions were as we described previously (21).

HPLC—Cells were plated onto poly-D-lysine-coated 12-well dishes to detect release of catecholamines. After stimulation, supernatant was collected and centrifuged (4 °C, 15,000 rpm, 5 min). The amount of catecholamines secreted into the medium was assayed using HPLC system (Bio Analytical System, Inc., West Lafayette, IN) equipped with an electrochemical detector (LC-44). Catecholamines were separated using a C-18 reversed phase column at a flow rate of 1 ml/min.

Detergent Solubilization and Sucrose Gradient Fractionation—Raft fractions were prepared as previously described (22) with minor modifications. Briefly, cells were washed twice with ice-cold PBS and once with 25 mM Mes, 150 mM NaCl, pH 6.5 (MBS) after stimulation. The cells were then resuspended in 2.5 ml of 1% Triton X-100 in MBS supplemented with protease inhibitors and phosphatase inhibitors and incubated at 4 °C for 20 min. The solubilized cells were homogenized with 10 strokes of a Dounce homogenizer, and 2 ml of the homogenate was added to an equal volume of 80% (w/v) sucrose in MBS. The solution was overlaid successively with 4 ml of 30% sucrose and 4 ml of 5% sucrose. After centrifugation (4 °C, 39,000 rpm, 18 h) in an SW41 rotor, 1-ml fractions were collected from the top of the gradient. The recovered gradient fractions were analyzed by immunoblotting with antibodies against several proteins as indicated.

Immunoprecipitation and Immunoblotting—Cell lysis and immunoblot analysis were performed as described previously (23) with minor modifications. Briefly, serum-starved cells were stimulated as indicated, washed with ice-cold PBS, and lysed using a hypotonic buffer (10 mM Tris/HCl, pH 7.5, 10 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 1% Nonidet P-40, and 0.25% sodium deoxycholate) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) and phosphatase inhibitors (1 mM Na3VO4 and 30 mM Na4O7P2). After sonication, samples were centrifuged at 15,000 x g for 15 min. Immunoprecipitation of the EGFR (24) and Src (25) was performed as previously described. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and the following procedures were performed as previously described (23). Bands were visualized with the ECL detection system.

Immunocytochemical Analysis—PC12 cells transfected with green fluorescent protein-tagged PLC{gamma}1 using LipofectAMINE were serum-starved for 24 h before use. Cells fixed with paraformaldehyde were incubated with blocking solution (2% bovine serum albumin and 100 µl/ml equine serum in PBS) for 1 h at room temperature. Cells were then incubated overnight at 4 °C with anti-flotillin-1 mAb, followed by incubation with rhodamine-conjugated anti-mouse IgG. Slides were mounted and visualized by confocal laser scanning microscopy (LSM510 Meta, Zeiss, Oberkochen, Germany).

Statistical Analysis—All experiments, including the immunoblots, were independently repeated a minimum of three times. All traces and immunoblots presented are representative of more than three separate experiments. All quantitative data are presented as means± S.E. Comparisons between two groups were analyzed via Student's t test, and values of p < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sensitization of EGF-induced Intracellular Calcium Increase ([Ca2+]i) by BK—EGF itself failed to cause any changes in the resting [Ca2+]i when administrated alone, but a substantial amount of [Ca2+]i increase was evoked by EGF after bradykinin pretreatment in both primary cultures of rat adrenal chromaffin cells and PC12 cells (Fig. 1). This "sensitization" of the EGF-induced calcium rise was highly specific to BK pretreatment, which could not be mimicked by UTP, an agonist of G protein-coupled P2Y2 purinoceptors, or other calcium-elevating agents such as high K+ and ionomycin (Fig. 2A). Sensitization of the EGF-induced [Ca2+]i increase occurred in a BK concentration-dependent manner (EC50, 4.9 ± 0.6 nM) and was completely blocked by HOE140, an antagonist of bradykinin B2 receptor (Fig. 2C), suggesting that the sensitization was specific to the activation of B2 receptor. The magnitude of the EGF-induced Ca2+ rise depended on EGF concentration with an EC50 value of 80.3 ± 17.1 pM (Fig. 2B). EGF concentration also affected the kinetics of the EGF-induced [Ca2+]i rise (Fig. 2D). Cells treated with higher concentrations of EGF required shorter time to reach the peak height compared with those treated with lower concentrations. BK induced a long lasting sensitization, which showed its maximal effect after 10 min of BK pretreatment and lasted up to 1 h (Fig. 2E).



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  		FIG. 1.
BK sensitizes EGF-induced [Ca2+]i increase. Rat adrenal chromaffin cells (A) and PC12 cells (B) were loaded with Fura-2/AM and subjected to microscopic fluorescent calcium imaging. 20 nM EGF and 1 µM BK was applied as indicated. Representative traces from at least five independent experiments are shown.

 



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  		FIG. 2.
Sensitization of the EGF-induced [Ca2+]i increase. Fura-2/AM-loaded PC12 cells (1 x 106) were stimulated as indicated. A, sensitization is specific to BK pretreatment. 20 nM EGF was treated ({blacktriangleup}) after treatment with 1 µM BK, 100 µM UTP, 70 mM KCl, or 1 µM ionomycin where indicated ({triangleup}). Shown are representative traces from four independent experiments. B, sensitization depends on EGF concentration. Various concentrations of EGF were treated either alone ({blacktriangleup}) or after BK treatment (1 µM, 5 min) (•). C, sensitization is dependent on B2 receptor activation. 20 nM EGF was treated after treatment with various concentrations of BK (•). Cells were exposed to HOE140 (3 µM) 3 min prior to BK stimulation ({blacktriangleup}). D, EGF concentration affects kinetics of EGF-induced [Ca2+]i increase. Various concentrations of EGF were applied after BK (1 µM, 5 min) treatment. Time to reach the peak value of EGF-evoked [Ca2+]i rise was measured. E, time course of the sensitization. 1 µM BK was applied for the indicated time periods, followed by 20 nM EGF. B, C, and E, peak heights of the EGF-induced [Ca2+]i increase were measured, and net increases in [Ca2+]i were calculated. Data presented in B-E are means ± S.E. values from at least three separate experiments.

 
EGF-evoked elevation of [Ca2+]i after BK treatment could be the result of either Ca2+ release from intracellular Ca2+ stores or Ca2+ influx from extracellular space. EGF-induced [Ca2+]i rise was completely blocked when cells were exposed to thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases, but was not significantly affected when extracellular Ca2+ was chelated with EGTA (Fig. 3, A and B). These results suggest that the EGF-induced [Ca2+]i rise sensitized by BK is mainly Ca2+ release.



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  		FIG. 3.
PLC-dependent Ca2+ release is induced by EGF after BK treatment. Fura-2/AM-loaded PC12 cells (1 x 106) were stimulated as indicated. A, [Ca2+]i rise evoked by EGF is from intracellular stores. 1 µM BK was treated followed by treatment with vehicle (a) or 1 mM EGTA (b and c) where indicated ({triangleup}), and then finally 20 nM EGF (a and b) or 70 mM KCl (c) was applied. [Ca2+]i was not elevated by KCl, confirming that extracellular Ca2+ was chelated under these conditions. Typical Ca2+ traces from four separate experiments are presented. B, using a similar experimental paradigm as A, cells were exposed to 1 mM EGTA, 1 µM thapsigargin, or vehicle after 1 µM BK treatment, and then stimulated with 20 nM EGF. C, sensitization is dependent on PLC. 3 µM U73343 [GenBank] (a) or U73122 [GenBank] (b) was applied where indicated ({triangleup}) after 1 µM BK treatment, then finally 20 nM EGF was treated. D, quantification of results from C. Quantification of data for B and D was achieved by expressing net [Ca2+]i increase in the peak value of the EGF-evoked [Ca2+]i rise as a percentage of control. Data presented are means ± S.E. values from three separate experiments.

 
We then examined the involvement of PLC in the sensitization. As shown in Fig. 3 (C and D), sensitization was completely blocked by U73122 [GenBank] , a selective inhibitor of PLC, whereas its inactive analog U73343 [GenBank] had no effect. Therefore, EGF-induced [Ca2+]i increase sensitized by BK was interpreted to reflect PLC-dependent Ca2+ release.

Involvement of EGFR in the Sensitization—To investigate the involvement of EGFR, we firstly tested the effect of tyrphostin AG1478, a specific inhibitor of EGFR kinase, on the sensitization. EGF-induced [Ca2+]i increase sensitized by BK was completely abolished by AG1478 (Fig. 4A), suggesting that the sensitization required EGFR kinase activity. We next monitored the level of tyrosine phosphorylation of EGFR. As shown in Fig. 4B, BK caused transactivation of the EGFR as previously reported (26). When EGF was treated 10 min after BK pretreatment, the level of EGFR tyrosine phosphorylation was synergistically enhanced compared with that stimulated with either BK or EGF alone (Fig. 4B). Together, these results suggest that phosphorylation and activation of the EGFR was critically involved in the sensitization.



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  		FIG. 4.
Involvement of EGFR in the sensitization. A, tyrosine kinase activity of EGFR is required. Fura-2/AM-loaded PC12 cells (1 x 106) were treated with 1 µM BK, followed by vehicle (a) or 500 nM AG1478 (b), where indicated ({triangleup}), and finally stimulated with 20 nM EGF ({blacktriangleup}). Typical Ca2+ traces from three separate experiments are presented. B, EGF-induced EGFR phosphorylation is potentiated by BK. PC12 cells were treated as indicated. NT, untreated; BK 2', 1 µM, 2 min; BK 10', 1 µM, 10 min; BK/EGF, 1 µM BK (10 min) followed by 200 pM EGF (2 min); EGF, 200 pM, 2 min. Cell lysates were subjected to SDS-PAGE after immunoprecipitation with anti-EGFR Ab. Tyrosine-phosphorylated EGFRs were detected by immunoblotting with anti-phosphotyrosine Ab (PTYR). The amounts of the precipitated EGFRs were monitored by reprobing the same blot with anti-EGFR Ab.

 
Role of Src Kinase in the BK-induced Sensitization of EGF Responses—Src family kinases have been proposed to participate in the communication between GPCRs and receptor tyrosine kinases (RTKs) as important mediators (27, 28). In addition, p60Src has been shown to be activated by BK (29). We thus, examined the effect of PP2, a selective inhibitor of Src kinase, on the sensitization. As shown in Fig. 5A, PP2 completely abolished the sensitization of EGF-induced [Ca2+]i rise. In addition, the sensitization of EGF-induced Ca2+ rise was inhibited by transient transfection of PC12 cells with the kinase-defective mutant of p60Src (SrcKM) (Supplemental Fig. 1), indicating that p60Src is critically involved. We also confirmed the requirement of Src kinase activation in the sensitization of EGF-induced [Ca2+]i increase in primary cultures of rat adrenal chromaffin cells (Fig. 5B).



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  		FIG. 5.
Involvement of Src kinase in the sensitization. A, PP2 abolishes the sensitization. Fura-2/AM-loaded PC12 cells (1 x 106) were treated with vehicle (a) or 10 µM PP2 (b) where indicated ({triangleup}) followed by 1 µM BK, and finally stimulated with 20 nM EGF. Typical Ca2+ traces from five separate experiments are presented. B, requirement of Src kinase activation in the sensitization in rat adrenal chromaffin cells. 10 µM PP2, 1 µM BK, and 200 pM EGF was applied to Fura-2/AM-loaded adrenal chromaffin cells as indicated. A representative trace from four independent experiments is shown. C, Src is involved in the potentiation of EGF-induced EGFR phosphorylation by BK. Left, PC12 cells were exposed to 10 µM PP2 (+) or vehicle (-) 3 min prior to any stimuli. Abbreviations are described in Fig. 4B. Right, cells were exposed to vehicle (-), 5 or 10 µM PP2 for 3 min, followed by 200 pM EGF. Detection of tyrosine-phosphorylated EGFRs (PTYR) and the EGFR was performed as in Fig. 4B. D, Src is involved in the sensitization of EGF-induced IP3 generation. PC12 cells were stimulated as indicated, and IP3 was measured as described under "Experimental Procedures." EGF, 20 nM, 1 min; BK/EGF, EGF (200 pM, 1 min) after BK (1 µM, 10 min). 10 µM PP2 ({blacksquare}) or vehicle ({square}) was treated 3 min prior to any stimuli. Data are presented as a percentage of control, which represents basal level of IP3 treated only with vehicle. Each bar represents a mean ± S.E. value from three separate experiments. **, p < 0.01.

 
To investigate the target site on which p60Src acts in the sensitization process, we examined the possible role of Src in activation of EGFR and PLC. Tyrosine phosphorylation of EGFR and phosphoinositide hydrolysis were monitored to indicate activities of EGFR and PLC, respectively. As shown in Fig. 5C (left), BK-induced transactivation of EGFR was almost completely blocked by PP2, suggesting that Src acts upstream of EGFR activation. Importantly, PP2 significantly inhibited the potentiation effect of BK on the subsequent EGF-induced EGFR phosphorylation, so that phosphorylation of EGFR was reduced to the level comparable to that stimulated with EGF alone. This was as expected, because tyrosine phosphorylation of the EGFR induced by EGF alone was not disturbed by PP2 (Fig. 5C, right). Together, these results suggest that Src is specifically involved in the BK-induced potentiation of EGFR phosphorylation, but not in the autophosphorylation of EGFR induced by EGF alone.

Inositol 1,4,5-trisphosphate (IP3) was not generated to a detectable amount by EGF alone (Fig. 5D). However, when EGF was applied after BK-induced IP3 generation returned to basal level, it gave rise to substantial amount of IP3 generation (206 ± 29 percentage of basal), which was completely blocked by PP2. These results suggest that Src is critically involved in the sensitization process by up-regulating EGFR-mediated activation of PLC.

Induction of EGF-mediated Exocytosis by BK—Calcium is an important factor in transmitter release in neurons and neuroendocrine cells (30). Because EGF became capable of inducing Ca2+ increase after BK pretreatment, we examined whether BK could also induce sensitization of neurotransmitter release evoked by EGF. The effects on transmitter release were determined by using carbon fiber amperometry and HPLC. EGF itself failed to induce exocytosis to a detectable amount under both techniques (Fig. 6). Accumulated results from several cells demonstrated that EGF became capable of eliciting substantial amount of exocytotic events when EGF was applied after BK-induced secretory events returned to basal level. HPLC analysis also revealed that BK pretreatment up-regulated the subsequent EGF-evoked dopamine secretion, which was completely inhibited by PP2 (Fig. 6B).



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  		FIG. 6.
Src-mediated sensitization of EGF-evoked exocytosis. A, EGF-induced exocytosis is sensitized by BK. Upper panel, typical amperometric responses from PC12 cells, following addition of 20 nM EGF and 1 µM BK as indicated are shown. Lower panel, Rate of exocytosis of the upper panel was normalized. Number of amperometric spikes over a 30-s recording period was divided by that over a 30-s control period. Each bar represents a mean ± S.E. value obtained from 8 to 15 cells pooled from 6 to 10 separate experiments on different batches of cells. The normalized rate of exocytosis in the control period is assigned a value of 1.0 and indicated by horizontal broken lines. B, Src is required for sensitization of EGF-induced secretion. PC12 cells were stimulated as indicated, and transmitter release was measured by HLPC. BK, 1 µM, 10 min; EGF, 20 nM, 10 min; BK/EGF, EGF (20 nM, 10 min) after BK treatment. 10 µM PP2 ({blacksquare}) or vehicle ({square}) was applied 3 min prior to any stimuli. Each bar represents a mean ± S.E. value from three separate experiments. **, p < 0.01.

 
Localization of Signaling in the Raft Fraction upon BK Stimulation—Historically, lipid rafts have been defined by their low density and relative resistance toward solubilization with Triton X-100 at 4 °C (31). This nonionic detergent extraction leads to the isolation of a light membrane fraction, which contains several membrane and cytoplasmic proteins enriched in lipid rafts (8, 32). We used this well-established protocol to purify rafts from PC12 cells. Flotillin, a structural component of lipid rafts (6, 22), but not BiP/GRP78 (an endoplasmic reticulum protein), was recovered in fraction 5.

It was found that EGFR and p60Src were enriched in the flotillin-containing light membrane fraction during density gradient centrifugation, properties that are consistent with their association with lipid rafts (Fig. 7A). As shown in Fig. 7B, BK caused tyrosine phosphorylation of EGFR in the raft fraction, but the level of EGFR and Src in the rafts was not affected. Interestingly, PLC{gamma}1, which was mostly found in the soluble fractions of unstimulated cells, became detergent-resistant after BK treatment. Confocal microscopy analysis also revealed a clear translocation of PLC{gamma}1 from cytosol to flotillin-enriched plasma membrane upon BK stimulation (Fig. 7C), suggesting recruitment of PLC{gamma}1 to the rafts.



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  		FIG. 7.
Phosphorylation and translocation events occur in the raft fraction. A, EGFR and c-Src, but not PLC{gamma}1, are enriched in the raft fraction. PC12 cell lysates were fractionated by sucrose density gradient centrifugation as described under "Experimental Procedures." Fractions were collected from the top, and aliquots from each fraction were resolved by SDS-PAGE and probed with the indicated Abs. B, fraction 5 collected from vehicle (NT)- or BK (1 µM, 10 min)-treated cells were subjected to SDS-PAGE and immunoblotted with the indicated Abs. Tyrosine-phosphorylated EGFR (P-EGFR) was detected by anti-phosphotyrosine Ab after immunoprecipitation with antibodies against EGFR. A and B, different lanes contain equal amounts of total proteins. C, confocal images demonstrate extensive colocalization of PLC{gamma}1 (green) and flotillin (red) upon BK stimulation (1 µM, 10 min) in the plasma membrane of PC12 cells.

 
We further investigated whether BK-induced phosphorylation of EGFR in the rafts occurs in a Src-dependent manner. For this purpose, first it was confirmed that phosphorylation of Src on Tyr-418, indicative of Src activation, occurred in the raft fraction upon BK stimulation (Fig. 8A). Importantly, BK caused phosphorylation of the EGFR on Tyr-845, a phosphorylation known to be dependent on the kinase activity of Src (18), in the raft fraction. PP2 completely inhibited this BK-induced EGFR phosphorylation on Tyr-845, as well as phosphorylation of Src on Tyr-418.



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  		FIG. 8.
Src-dependent EGFR phosphorylation on Tyr-845, which occurs in the rafts, is critical to the sensitization. A, PC12 cells were exposed to vehicle (-) or 5 µM of PP2 (+) for 3 min and then stimulated with vehicle (NT) or BK (1 µM, 10 min) in the presence or absence of PP2. Raft fractions collected from each sample were subjected to SDS-PAGE and immunoblotted with the indicated Abs. B, transfection with a Y845F mutant of EGFR inhibits the sensitization. PC12 cells transfected with the mutant of EGFR (EGFR Y845F) or vector alone were loaded with Fura-2/AM for Ca2+ measurements. Cells were stimulated with EGF (20 nM, 5 min) after BK treatment (1 µM, 5 min). Quantification of data was achieved by expressing the EGF-evoked [Ca2+]i rise in mutant-transfected cells as a percentage of that in vector-transfected cells. Each bar represents a mean ± S.E. value from four separate experiments. **, p < 0.01.

 
Involvement of Src-dependent EGFR Phosphorylation in the Sensitization—We then examined the effect of a variant receptor harboring a Y845F mutation in the EGFR. We observed that expression of this mutated receptor did not significantly alter BK-induced [Ca2+]i increase, but inhibited the subsequent EGF-induced Ca2+ rise by 32 ± 5% (Fig. 8B), which corresponded to the transfection efficiency of the plasmids (30 ± 7%). These results suggest that Src-dependent phosphorylation of the EGFR on Tyr-845 is crucial to the sensitization.

Effect of Cellular Cholesterol Depletion and Add-back in the Sensitization—Lipid raft microdomains are enriched in cholesterol, which appears to be necessary to maintain their integrity (8). To determine whether the integrity of rafts is required for the maintenance of sensitization, we used methyl-{beta}-cyclodextrin (M{beta}CD), which reduces the cholesterol content of the cell, leading to disruption of the raft structures (33-35). As shown in Fig. 9A, M{beta}CD disrupted flotillin-1 location in the buoyant fraction of sucrose gradients, in comparison to the normally restricted distribution. There was also a reduction in the quantity of EGFR and Src within the raft fraction. Similarly, a reduction of phosphorylated EGFR and Src levels was observed. BK-induced recruitment of PLC{gamma}1 and B2 receptor to the rafts was also inhibited by M{beta}CD. Furthermore, sensitization of the EGF-induced Ca2+ rise was completely abrogated (Fig. 9B), suggesting that the integrity of lipid rafts is required to maintain the receptor cross-talk. However, neither high K+- nor carbachol-mediated Ca2+ rise was inhibited by lipid raft dispersion, excluding the possible nonspecific toxic effects of M{beta}CD.



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  		FIG. 9.
Effects of cellular cholesterol depletion and add-back on the sensitization. A, effects of M{beta}CD on association of signaling proteins with rafts. Upper panel, control and M{beta}CD (5 mM, 37 °C, 30 min)-treated PC12 cells were fractionated by sucrose density gradient centrifugation. Fractions were subjected to SDS-PAGE and immunoblotted with anti-flotillin-1 Ab. Lower panel, raft fractions collected from control and M{beta}CD-treated cells stimulated with vehicle (NT) or BK were probed with Abs as indicated. B, PC12 cells incubated with vehicle (a) or M{beta}CD (b) were loaded with Fura-2/AM for Ca2+ measurements. Upper panel, 1 µM BK was applied followed by 20 nM EGF. Typical Ca2+ traces from four separate experiments are presented. Lower panel, net [Ca2+]i increase evoked by agonist of interest in M{beta}CD-treated cells is presented as a percentage of that in vehicle-treated cells. BK/EGF, 20 nM EGF-induced [Ca2+]i rise after BK treatment; KCl, 70 mM; carbachol, 1 mM. C, cholesterol add-back restores the inhibitory effect of M{beta}CD. PC12 cells were incubated with either vehicle (Ctrl) or M{beta}CD (M{beta}CD and M{beta}CD/Chol). The medium of one M{beta}CD-treated culture was removed and replaced with 0.2 mM cholesterol complex with M{beta}CD for 30 min at 37 °C (M{beta}CD/Chol). Cells were loaded with Fura-2/AM for Ca2+ measurements. EGF-induced [Ca2+]i rise after 1 µM BK stimulation was measured and presented as a percentage of that in vehicle-treated cells. B and C, each bar represents a mean ± S.E. value from three separate experiments. D, integrity of rafts is required in the sensitization process in adrenal chromaffin cells. Cholesterol was depleted from and added back to rat adrenal chromaffin cells as described in C. Cells were loaded with Fura-2/AM and subjected to microscopic fluorescent calcium imaging. Cells were treated with 1 µM BK, 20 nM EGF, and 70 mM KCl as indicated. Typical traces from three independent experiments are shown.

 
Whereas M{beta}CD can remove cholesterol from cell membranes, soluble complexes of cholesterol and M{beta}CD can mediate the incorporation of cholesterol into membranes (13, 35). As shown in Fig. 9C, M{beta}CD-induced loss of sensitization was restored by treatment with cholesterol-M{beta}CD complexes. Disruption of sensitization by cholesterol depletion and restoration by cholesterol add-back were also observed in rat adrenal chromaffin cells (Fig. 9D). These results confirm that the effect of M{beta}CD was due to its capacity to remove cholesterol from cells rather than by some other possible nonspecific toxic effects. These findings suggest that BK-induced sensitization of EGF signaling requires the integrity of cholesterol-enriched domains and imply that localization of protein components to these low density domains might be necessary for the proper function of this signaling system.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cross-communication between heterologous cellular signaling systems has now emerged as a common principle emphasizing that most biological responses result from the functional integration of specific and diverse network of intracellular signaling pathways (36). Although initial studies suggested that signaling pathways triggered by GPCRs were dissociated from RTK-mediated signaling events, it has become increasingly evident that there is cross-communication between the signaling systems of these two major classes of cell surface receptors (37). One of the most extensively studied examples is the activation of the mitogen-activated protein-kinases (MAPKs) upon GPCR stimulation. A host of distinct signaling pathways contribute to link GPCRs to the MAPK cascade depending on the type of the cell as well as the receptor. Rather than conflicting, multiple examples stress the importance of the cellular context when defining cross-talk between heterologous signal transduction pathways (28). In contrast to the massive literature describing activation of the MAPK pathway upon GPCR stimulation, interaction of GPCRs and RTKs at the level of PLC activation and intracellular calcium control has been investigated to a much smaller extent. In the present study we demonstrate that EGFR-mediated PLC activation, [Ca2+]i increase and neurotransmitter release are sensitized specifically in response to B2 receptor stimulation. This study provides evidence that p60Src plays an essential role and that maintenance of the integrity of cholesterol-rich membrane lipid rafts is required in this sensitization process, providing a novel insight into the signaling network involving two major classes of the cell surface receptors.

The term "sensitization" we use throughout the text to describe EGF-induced signaling events after BK pretreatment is different from transactivation termed by Ullrich's group (38). In contrast to the GPCR-induced transactivation, which is independent of exogenous RTK agonists, BK-induced sensitization is observed only upon the addition of exogenous EGF. Sensitization we describe is highly specific to BK and cannot be induced by other calcium-elevating agents that have been reported to cause EGFR transactivation in PC12 cells (26, 39).

Interestingly, sensitization of the EGF-mediated cellular responses was specific to the calcium signaling pathway, i.e. the extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway was not affected by prior activation of bradykinin (Supplemental Fig. 2). Our results are consistent with those presented by Schlessinger and co-workers (40), who recently applied a genetic approach to analyze the roles played by EGFR and Src in signal transduction from GPCRs to the ERK/MAPK pathway. They have demonstrated the requirement of Src in the GPCR-induced phosphorylation of EGFR. By contrast, both Src and EGFR seemed to be dispensable for activation of the ERK/MAPK signaling cascade upon GPCR stimulation (40). Our results give consequence to the enhanced phosphorylation of the EGFR mediated by Src in response to BK stimulation, i.e. the Src-dependent phosphorylation of the EGFR sensitizes the EGF-mediated calcium signaling pathway, which results in neurotransmitter release.

EGF-induced increase in amperometric spikes was detected after ~90 s of EGF stimulation (Fig. 6A), at which EGF-induced [Ca2+]i rise reached its peak height (Fig. 2D). Accumulation of Ca2+ may have directly affected the exocytotic trigger (41), contributed to recruitment of and release from functionally distinct secretory pools (42, 43), or fulfilled threshold requirements for Ca2+ (43-46). In addition to the reasons mentioned above, it is also plausible to suggest that the increased exocytosis requires further steps. The precise mechanism, however, responsible for this delayed transmitter release remains to be determined.

BK-induced sensitization of the EGF-evoked [Ca2+]i rise required EGFR kinase activity, and BK significantly potentiated EGF-induced EGFR tyrosine phosphorylation (Fig. 4), suggesting that phosphorylation and activation of the EGFR is an important step in the sensitization. However, EGFR tyrosine phosphorylation per se does not fully contribute to the EGF-induced calcium response, because EGF alone induced strong phosphorylation of the EGFR but did not lead to increase in [Ca2+]i. Here we suggest that Src-dependent phosphorylation and activation of the EGFR in response to BK stimulation is crucial to the sensitization process. In many studies reporting cross-talk between GPCR and RTK, the mechanism by which GPCR stimulation can evoke activation of the RTK pathway is centered on the mediation of Src kinase (47, 48). The mediator role of Src may be direct or indirect, but the evidence so far suggests that Src is able to associate with and directly phosphorylate the EGFR (47). EGFR autophosphorylates at five known tyrosine residues upon EGF stimulation. Phosphorylations on the EGFR that are dependent on Src have been identified as Tyr-845 and Tyr-1101, which are not autophosphorylation sites. Interestingly, Parsons and colleagues reported that phosphorylation of these residues was associated with the formation of a heterocomplex between Src and activated EGFR and an enhanced phosphorylation of receptor substrates. including PLC{gamma} (18, 19, 49). Therefore, one mechanism by which Src could have synergy with the EGFR is by physically complexing with it and mediating the phosphorylation of non-autophosphorylation tyrosine residues, which in turn may result in hyperactivation of the receptor (48). In the present study we show that BK causes activation of p60Src, Src-dependent phosphorylation of the EGFR on Tyr-845 and recruitment of PLC{gamma}1 to the rafts where the EGFR is located (Figs. 7 and 8). Thus, it is plausible that BK might have induced a certain change in EGFR activity or structure via Src, thereby being capable of initiating the subsequent downstream events. One possible mechanism could be that EGFR phosphorylation mediated by Src might have resulted in enhanced recruitment and activation of PLC{gamma}1. Another possibility is that Src may act directly on PLC{gamma}1 or on a certain regulatory factor involved in PLC{gamma}1 activation in addition to acting on EGFR, because Src not only can act upstream of EGFR (27, 47) but can also be a regulator of PLC{gamma} (50-52).

Our results show that cholesterol depletion did not significantly affect the BK-induced Ca2+ increase, but completely abrogated the subsequent EGF-induced Ca2+ rise (Fig. 9). It seems that raft structures are not required in the early signaling of the BK receptor, but are necessary for the later sensitization process. Our present results are reminiscent of a recent study demonstrating that cholesterol depletion inhibited angiotensin-induced transactivation of the EGFR without affecting upstream signaling events such as angiotensin-induced [Ca2+]i increase (53).

Studies have suggested that BK receptors translocate to cholesterol-rich signaling domains upon stimulation (54-58). Interestingly, a previous study has provided evidence that the B2 receptor complex, including the B2 receptor and the associated G proteins, migrates from non-raft to raft locations in the plasma membrane upon agonist stimulation (54). Although how Src is activated by BK remains to be determined, it is plausible to suggest that activation of Src in the raft fraction might have occurred by the BK receptor complex translocated to the raft. Following activation of Src, Src-dependent phosphorylation of the EGFR occurs. When PLC{gamma}1 is recruited to the rafts where activated EGFRs are located, IP3 might be readily generated, because rafts are enriched in phosphatidylinositol 4,5-bisphosphate (57), the substrate of PLC. Generation of IP3 might then trigger Ca2+ release from intracellular calcium stores probably via activation of nearby IP3 receptors (Fig. 10). Compelling evidence suggests that plasma membrane receptors and IP3 receptors are organized together in a signaling microdomain that links the plasma membrane structure to the endoplasmic reticulum (2). Investigating localization of the IP3 receptors responsible for the sensitization as well as imaging local IP3 and Ca2+ signals upon stimulation might be of great interest and would give further insights into the sensitization mechanism.



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  		FIG. 10.
Suggested model for the BK-induced sensitization of EGF signaling. In resting state, EGFR and Src, but not PLC{gamma}1, are enriched in lipid rafts. BK stimulation might cause translocation of the B2 receptor complex to the rafts, where Src and EGFR are located. Upon BK stimulation, Src and EGFR become phosphorylated and activated in the rafts, and recruitment of PLC{gamma}1 to rafts occurs. Assemblage of EGFR, Src, and PLC{gamma}1 within a signaling microdomain might form a concentrating platform for the EGFR, thereby permitting specific receptor cross-talk and rendering precise modulation of the downstream signaling events.

 
The functional significance of lipid microdomains in cell biological processes is just beginning to be unraveled. The fundamental principle by which rafts exert their functions is a separation or concentration of specific membrane proteins and lipids in membrane microdomains (32, 58). Localizing and clustering signaling molecules on the cell surface at and within the appropriate site are critical for triggering and propagating signal transduction. In conclusion, we provide evidence that the receptors and effector molecules form a functional signaling complex in lipid rafts of neuroendocrine cells. Localizing EGFR, Src, and PLC{gamma}1 within a specialized microdomain of the plasma membrane following activation of the B2 receptor might provide a mechanism to expedite selective and efficient signal propagation by forming a concentrating platform that leads to the regulation of neuronal excitability.


   FOOTNOTES
 
* This work was supported by grants from the National Research Laboratory Program (2000-N-NL-01-C-150), the Brain Neurobiology Research Program (M10108000013-02B2300-00710) of the Ministry of Science and Technology (MOST), the Korea Science and Engineering Foundation (KOSEF; R01-1999-000-00145-0), the IMT2000 Fund from the Ministry of Health and Wellfare, and the Brain Korea 21 program of the Ministry of Education. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1 and S2. Back

{ddagger} To whom correspondence should be addressed. Tel: 82-54-279-2297; Fax: 82-54-279-2199; E-mail: ktk{at}postech.ac.kr.

1 The abbreviations used are: GPCR, G protein-coupled receptor; EGF, epidermal growth factor; EGFR, EGF receptor; BK, bradykinin; PLC, phospholipase C; Fura-2/AM, Fura-2 pentaacetoxymethyl ester; mAb, monoclonal antibody; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine; IP3, inositol 1,4,5-trisphosphate; RTK, receptor tyrosine kinase; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; Mes, 4-morpholineethanesulfonic acid; M{beta}CD, methyl-{beta}-cyclodextrin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Sarah J. Parsons for providing Y845F EGFR cDNA. We also thank Drs. Tadashi Yamamoto and Hiroshi Nishina for providing Src expression vectors.



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