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Differential Ca2+ signaling induced by activation of the epidermal growth factor and nerve growth factor receptors.

Stimulation by epidermal growth factor (EGF) of NIH3T3 cells overexpressing the EGF receptor (EGFR) results in a release of Ca2+ from internal stores. Ca2+ release is followed by an influx of extracellular calcium which can be recorded by the influx of the calcium surrogate Mn2+. Both Ca2+ release and Mn2+/Ca2+ influx are inhibited by expression of the dominant negative Asn17-Ras mutant and abrogated by microinjected neutralizing anti-Ras antibody Y13-259, whereas microinjection of the anti-Ras antibody Y13-238 which does not interact with the effector binding domain of Ras is without any effect on the EGF-induced Ca2+ transient. Neither Asn17-Ha-Ras nor the Y13-259 antibody interferes with the thapsigargin-induced Mn2+/Ca2+ influx. The nerve growth factor receptor (Trk)-mediated Ca2+ transient was found to be unaffected by the dominant negative Ras mutant or microinjected neutralizing anti-Ras antibodies. Substitution of the phospholipase Cγ1 (PLCγ1) binding site of the EGFR by the PLCγ binding domain of Trk renders the EGFR-induced Ca2+ influx insensitive to the expression of Asn17-Ha-Ras, whereas the Ca2+ signal induced by Trk carrying the PLC binding site of EGFR is Ras-dependent and abrogated by the dominant negative Ras mutant. It is concluded that the Ca2+ transient induced by the activated EGFR, not, however, the Ca2+ transient elicited by the activated NGFR/Trk, is a Ras-mediated phenomenon and that the role of Ras in regulating EGFR-induced Ca2+ influx depends on the structure of the PLCγ binding domain.

In previous reports, we and others had demonstrated that expression of transforming Ha-Ras leads to an enhanced Ca 2ϩ influx (5,(7)(8)(9)(10)(11). It remained unclear, however, whether this effect represents a gain-of-function of the mutated Ras protein or whether cellular Ras is also involved in regulating Ca 2ϩ entry mechanisms operating in nontransformed cells stimulated by growth factors which activate Ras-dependent signaling pathways.
In order to address this question, Ca 2ϩ transients induced by activation of epidermal growth factor receptor (EGFR) 1 and nerve growth factor receptor (NGFR/Trk) were investigated.
Both receptors are known to activate Ras and to cause an elevation of cytosolic free Ca 2ϩ (1,(12)(13)(14). The studies presented here were performed with NIH3T3 cells overexpressing either EGFR or an EGFR/Trk chimeric receptor consisting of an extracellular EGFR domain and the cytosolic domain of Trk as described previously (1). This system permits the study of both receptors in the same cellular environment employing the same agonist. In PC12 cells under physiological conditions, EGFR and NGFR induce opposite effects, activation of EGFR elicits proliferation, whereas stimulation of NGFR leads to differentiation (15)(16)(17)(18)(19). When expressed in fibroblasts, however, activation of NGFR causes a proliferative response (20). Both receptors have been shown to activate a phosphatidylinositol-specific phospholipase C, resulting in the generation of inositol 1,4,5-trisphosphate (IP 3 ) and a release of Ca 2ϩ from intracellular stores (1). In fibroblasts, Ca 2ϩ -release is followed or accompanied by an enhanced influx of Ca 2ϩ through voltageindependent Ca 2ϩ channels of the plasma membrane (21,22). Although the Ca 2ϩ transients elicited by activation of different receptor tyrosine kinases appear to be rather similar, the underlying mechanisms have been shown to differ in a receptorspecific fashion (23). These differences may result from variations in the contribution of Ca 2ϩ influx to the total Ca 2ϩ transient and the Ca 2ϩ channels involved. The data presented here demonstrate that the Ca 2ϩ influx following activation of EGFR is blocked by expression of a dominant negative (Asn 17 ) mutant of Ha-Ras and abrogated by microinjection of antibodies interacting with the effector binding domain of Ras whereas the Ca 2ϩ influx elicited by an activation of Trk is not affected by Asn 17 -Ha-Ras or anti-Ras antibody and appears to proceed by a Ras-independent mechanism. In an attempt to identify the mechanism responsible for the differences in signaling by the two receptor types, it was investigated whether the significantly different affinities to phospholipase C␥1 (PLC␥1) described in a preceding publication (1) are related to the distinct signal transmission. The studies revealed that an exchange of the phospholipase C␥1 (PLC␥1) binding sites between Trk and EGFR renders the Trk-induced Ca 2ϩ influx Ras-dependent and abolishes the Ras dependence of the EGFR-mediated Ca 2ϩ influx.

EXPERIMENTAL PROCEDURES
Materials-Fura-2/AM was obtained from Molecular Probes; culture media and sera were from Boehringer Ingelheim Bioproducts; SK&F96365 was from Smith Kline Beecham Pharmaceuticals; EGF and nifedipine were purchased from Sigma; pRSV-Asn 17 -Ha-Ras was kindly provided by L. de Vries, Laboratory for Physiological Chemistry, University of Utrecht, and pEF-neo GFP-S65T by S. Geley, Institute of Pathology, University of Innsbruck.
Cells-NIH3T3 fibroblasts overexpressing EGFR (EGFR6), NIH3T3 cells expressing a chimeric EGFR/Trk (ETR2), and NIH3T3 cells expressing the mutant receptors EGF-R.X2 or ET-R.X3 were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum and 2 mM L-glutamine at 37°C in a humidified atmosphere (95% air, 5% CO 2 ). The generation and characteristics of NIH3T3 transfectants expressing the mutant receptors ET-R2, EGF-R.X2, or ET-R.X3 had been described in a preceding publication (1). EGF-R.X2 carries the Trk residues 780 -790 which had been identified as the PLC␥1 binding site of Trk, in place of the EGFR amino acids 987-997. In ET-R.X3, Trk residues 780 -790 were replaced by the EGFR sequence 987-997 which had been characterized as a PLC binding site of EGFR. All cell lines exhibit similar expression levels for the receptors. Biological properties of these cell lines had been described previously (1).
Transient Transfection of Asn 17 -Ha-Ras and GFP-EGFR6 and ET-R2 fibroblasts were transfected with circular plasmid DNA (50 g/ml) by microinjection of single cells. The pipette solution contained 4 parts of pRSV dominant negative Asn 17 -Ha-Ras (2) or empty pRSV vector and 1 part of modified Aequorea green fluorescence protein (GFP-S65T in a pEF-neo vector) (3). 24 h after transfection, GFPpositive cells were investigated for [Ca 2ϩ ] i measurements and fura-2 fluorescence quench by Mn 2ϩ .
[Ca 2ϩ ] i Measurements-The cells (10 4 /ml) were plated on coverslips (diameter 22 mm) in 35-mm dishes (6-well plates) and cultured for 1 day. Loading with fura-2 was performed by incubation with 1 M fura-2/AM for 15 min. Then the cells were washed with HEPES buffer (HBS: 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , 5.5 mM glucose, 20 mM HEPES/NaOH, pH 7.4) and kept in HEPES buffer at room temperature. For determination of the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ) employing a single cell imaging system (Magical, Applied Imaging, Sunderland SR53HD, UK, Nikon Diaphot microscope), the coverslip was placed into the recording chamber and the cells were kept in nominally Ca 2ϩ -free HEPES buffer (HBS without Ca 2ϩ ) for 5 min. Where indicated, the cells were stimulated with 50 ng/ml EGF or 1 M thapsigargin. After 1 min, 1 mM CaCl 2 was added. The cytoplasmic Ca 2ϩ concentration was calculated from the ratios of background subtracted images (excitation wavelength 340 and 380 nm, emission wavelength 510 nm) according to the calibration procedure and equations described by Grynkiewicz et al. (4).
Fura-2 Fluorescence Quench by Mn 2ϩ -This quench was measured as described previously (5) according to the procedure of Kass et al. (6). Background subtracted single-cell images (excitation 360 nm, emission 510 nm) were captured, and the fura-2 quench by Mn 2ϩ was calculated as the percentage of fluorescence decrease of the initial fura-2 fluorescence 60 s after addition of 100 M Mn 2ϩ (final concentration). The initial fluorescence after fura-2 loading was similar in all cells under study (50 Ϯ 10 arbitrary units/cell).

The Ca 2ϩ Transient Elicited by the Activated EGF Receptor
Is Ras-dependent- Fig. 1A shows a representative single-cell recording of an EGF-induced Ca 2ϩ signal in EGFR6 cells. In order to discriminate between Ca 2ϩ release and Ca 2ϩ influx, cells were initially kept in Ca 2ϩ -free medium. Addition of EGF leads to a release of Ca 2ϩ from internal stores. After store depletion, Ca 2ϩ was added to the medium. This results in a second peak of intracellular Ca 2ϩ representing Ca 2ϩ influx and release of refilled stores. For a more sensitive determination of calcium influx, the Ca 2ϩ surrogate Mn 2ϩ was employed, and the quench of fura-2-loaded cells by exogenous Mn 2ϩ was determined in a single-cell imaging system. As shown in Table I, this EGF-induced Mn 2ϩ influx is sensitive to the Ca 2ϩ channel blockers SK&F96365 and La 3ϩ , but insensitive to nifedipine up to concentrations of 10 M. These results were likewise obtained by whole-cell patch clamp studies (data not shown).
In order to determine the role of Ras in the EGF-induced Ca 2ϩ transient, cells were transiently transfected with an expression plasmid encoding the dominant negative Asn 17 -Ras mutant. Transfection was performed by microinjection, and the transfected cells were identified by co-transfection with a vector encoding a mutated version of the green fluorescent protein (GFP-S65T). As shown in Fig. 1 and Table II, Asn 17 -Ras inhibits the EGF-induced Ca 2ϩ transient by interfering with both Ca 2ϩ release and Ca 2ϩ influx. Whereas Ca 2ϩ release is strongly but incompletely suppressed (Fig. 1A), Ca 2ϩ /Mn 2ϩ influx is completely abrogated (Table II). The results obtained with the dominant negative Ras mutant were confirmed by microinjection of anti-Ras antibodies. The monoclonal antibody Y13-259 had been shown to interact with the effector binding domain of Ras and to inhibit the proliferative effect of serum growth factors (24). As shown in Table III, microinjection of this antibody completely inhibits the EGF-induced Ca 2ϩ /Mn 2ϩ influx in EGFR6 cells. Microinjection of the non-neutralizing anti-Ras antibody Y13-238 (25) did not affect Ca 2ϩ /Mn 2ϩ influx (Table  III). Microinjected unspecific fluorescein isothiocyanate-labeled antibody which was used to identify the treated cells also did not interfere with the Ca 2ϩ influx (data not shown).
Thapsigargin-induced Ca 2ϩ Influx Is Not Ras-dependent-The absolute Ras dependence of the EGF-induced Ca 2ϩ influx raised the question whether Ras is involved in regulating the activity of store-operated calcium channels. For this reason, intracellular Ca 2ϩ stores were depleted with thapsigargin, and the effects of an expression of the dominant negative Ras mutant and microinjected, neutralizing anti-Ras antibody were determined. The data revealed, however, that neither Asn 17 -Ras nor the neutralizing anti-Ras antibody are able to interfere with thapsigargin-induced store-regulated Ca 2ϩ influx (Tables  II and III).
The Ca 2ϩ Transient Induced by Activated NGFR/Trk Is Ras Independent-The role of Ras in the NGFR/Trk-induced Ca 2ϩ signal was studied in ETR2 cells. These cells represent NIH3T3 fibroblasts expressing a chimeric EGFR/Trk consisting of the extracellular EGF binding domain of the EGFR and the cytosolic region of Trk (1). Activation of Trk by EGF results in a calcium signal which resembles the Ca 2ϩ transient observed after activation of EGFR in EGFR6 cells (Fig. 1B and Table I).
In contrast to the EGFR-induced Ca 2ϩ signal, neither release nor influx of Ca 2ϩ are affected by an expression of Asn 17 -Ras or microinjected neutralizing anti-Ras antibodies (Fig. 1B, Tables  II and III).
The Ras Dependence of the Receptor-mediated Ca 2ϩ Influx Is Determined by the Structure of the Phospholipase C␥1 (PLC␥1) Binding Domains-A major difference between EGFR and NGFR/Trk is the affinity to PLC␥1. Compared to the EGFR, the affinity of Trk to PLC␥1 is approximately 100-fold higher (26). The high affinity of activated Trk was shown to be defined by Ϯ5 amino acid residues flanking phosphorylated tyrosine 785. Changing the tyrosine at this position to phenylalanine causes a PLC␥ binding-incompetent mutant receptor that cannot induce any IP 3 or Ca 2ϩ signal upon EGF stimulation (1). In the EGFR, a PLC␥1 binding site surrounding tyrosine 992 had been identified. In addition to the EGFR domain surrounding Tyr 992 , which exhibits the highest affinity for PLC␥, secondary low affinity binding sites such as Tyr 1068 and Tyr 1173 could be characterized (27). In order to investigate the significance of the distinct binding properties for the Ras dependence of the Ca 2ϩ signal, exchange mutants were employed. The exchange mutant EGF-RX carried the Trk residues 780 -790 in place of the EGFR amino acids 987-997; in ET-R, Trk residues 780 -790 were replaced by the EGFR sequence 987-997, to yield ET-RX as described previously (1). The expression levels and functionality of the mutant receptors were determined in stably transfected NIH3T3 cells as described before (1). The ability of the mutant receptors to activate Ras independently of their PLC␥ binding domain was ascertained (data not shown). As shown in Fig. 2, substitution of the PLC␥1 binding site of Trk by the PLC␥ binding domain of the EGFR renders the Ca 2ϩ influx into cells overexpressing the mutant Trk receptor (ET-R.X3) sensitive to dominant negative Ras or to microinjected neutralizing anti-Ras antibodies (Table III). Inversely, the Ca 2ϩ influx elicited by the activated EGFR which is completely blocked by Asn 17 -Ras becomes refractory to the expression of the dominant negative Ras mutant if the exchange mutant EGF-R.X2 carrying the PLC␥ binding site from Trk is expressed (Fig. 2). Thus, the Ras dependence of the Ca 2ϩ influx induced by the activated EGFR or Trk, respectively, is determined by the structure of the PLC␥ binding sites and can be transferred from one receptor to the other together with the corresponding binding domain of the receptors. DISCUSSION The data presented here demonstrate that the Ca 2ϩ signal induced by an activation of the epidermal growth factor receptor (EGFR) is mediated by a Ras-dependent mechanism. The complete inhibition of Ca 2ϩ influx which is seen in cells expressing the dominant negative Asn 17 -Ras mutant or observed after microinjection of neutralizing anti-Ras antibodies is prob-ably due to the suppression of Ca 2ϩ release (Fig. 1A). The attenuated depletion of internal Ca 2ϩ stores may be insufficient to activate a store-operated Ca 2ϩ influx. That Ras is not required for the activation of store-operated Ca 2ϩ channels of the plasma membrane is supported by the insensitivity of the thapsigargin-mediated Ca 2ϩ influx to Asn 17 -Ras or microinjected neutralizing anti-Ras antibodies. The mechanism by which Ras regulates Ca 2ϩ release remains to be elucidated. According to a recent publication, the EGF-induced Ca 2ϩ transient is completely abolished by dominant negative Asn 17 -Rac1 (23) indicating that Asn 17 -Rac1 also interferes with Ca 2ϩ release; otherwise, the initial rise in cytosolic free Ca 2ϩ release would have been unaffected. These findings together with the data reported here suggest that the EGF-induced Ca 2ϩ release involves Ras and Rac1. The activation of c-Jun amino-terminal kinases (JUNKs) by EGF has also been shown to require Ras and Rac1 (28,29). Thus, the activated EGFR may employ a similar pathway for the generation of the Ca 2ϩ signal and the activation of JUNK. The release of internal Ca 2ϩ is usually mediated by inositol 1,4,5-trisphosphate (IP 3 ) generated by a phospholipase C (PLC) (30). Ligand-activated EGFR is known to bind and activate PLC␥ (31). Although evidence for Ras as an upstream effector or regulator of PLC␥ has been presented (32,33), the detailed mechanism by which Ras could regulate PLC activity is still unclear. Recently, it has been shown that the SH2 domains of p120 Ras-GAP exhibit a similar affinity to a binding site of the EGFR as the SH2 domains of PLC␥ (34). The region around the phosphorylated tyrosine at position 992 was also described as a high affinity binding site for protein-tyrosine phosphatase 1b (PTP1b) and PLC␥ as well as GAP are equally effective in competing with PTP1b for binding to the TABLE I EGF-induced Ca 2ϩ influx determined by Mn 2ϩ quenching of fura-2 EGF-R6 and ET-R2 fibroblasts were loaded with fura-2. The Ca 2ϩ influx was estimated from Mn 2ϩ -dependent fura-2 quench detected 60 s after addition of 100 M MnCl 2 (final concentration). After preincubation with the calcium channel blockers SK&F96365, LaCl 3 , or nifedipine in the indicated concentrations for 1 min, the cells were stimulated with EGF. The fura-2 quench was calculated as the percentage of fluorescence decrease of the initial fura-2 fluorescence. The values represent the percentage of the induced Mn 2ϩ quench (mean Ϯ S.E. (8 Յ n Յ 16)).  (Asn 17 -Ha-Ras) on Ca 2ϩ influx EGF-R6 and ET-R2 fibroblasts were transiently transfected with the dominant negative Asn 17 -Ha-Ras construct or the empty control vector and cotransfected with the green fluorescence marker construct by microinjection as described under "Experimental Procedures." 24 h after transfection, the cells were loaded with fura-2, and the Ca 2ϩ influx was estimated from Mn 2ϩ -dependent fura-2 quench. Where indicated, the cells were stimulated with EGF and thapsigargin for 1 min. The fura-2 quench was calculated as the percentage of fluorescence decrease of the initial fura-2 fluorescence.    EGFR (35). Thus, Ras-GAP and PLC␥ may compete for the same binding site of the EGF receptor. Since Ras-GAP also binds to the effector domain of GTP-charged Ras (36), which requires a release from the receptor (37), conditions which favor the accumulation of Ras-GDP-like expression of Asn 17 -Ras (38,39) or blockade of the effector binding domain of Ras by a neutralizing antibody may enhance the association of Ras-GAP to the PLC␥ binding site of the EGFR and thereby reduce the activation of PLC␥. This competition may be more relevant for the EGFR than for Trk as (i) the affinity of PLC␥ to the binding site around tyrosine 992 of the EGFR is significantly lower than the affinity of PLC␥ to Trk where residues flanking tyrosine 785 form a high affinity binding domain for PLC␥ (1), (ii) there is so far no evidence for a competition between GAP and PLC␥ for a common binding site on Trk. The assumption that the Ras dependence of the Ca 2ϩ influx induced by the activated receptors is correlated to their affinities for PLC␥1 could be confirmed by studies employing receptor mutants. We had shown previously (1) that the EGF-induced IP 3 response in cells overexpressing the EGFR/Trk (ETR) is proportional to the affinities of PLC␥1 to the corresponding receptors, i.e. high for ET-R cells and low for EGF-R cells. Substitution of tyrosine at position 785 of the ETR by a phenylalanine eliminates binding of PLC␥1 to the ETR and abrogates the EGFinduced IP 3 and Ca 2ϩ signals (1). Exchange of the PLC␥1 binding sites of EGFR and Trk reduces the binding of PLC␥1 to the mutated ETR and enhances PLC␥1 binding to the EGFR exchange mutant. Accordingly, EGF-induced IP 3 and Ca 2ϩ signals were found to be strictly proportional to the binding affinities of PLC␥1 to the corresponding receptor mutants (1).
Activation of a mutant EGFR carrying the PLC␥1 binding domain of Trk instead of the endogenous PLC␥ binding domain leads to a Ca 2ϩ influx which is unaffected by dominant negative Ras, although the expression levels of the wild type EGFR6 and the mutant EGF-R.X2 were found to be similar. Further-more, the Trk-induced Ca 2ϩ influx which was found to be unaffected by dominant negative Ras, becomes Ras-dependent if the PLC␥1 binding site of Trk is replaced by the PLC binding domain of the EGFR. The data obtained with the exchange mutants also demonstrate that the insensitivity of the Trkinduced Ca 2ϩ influx to dominant negative Asn 17 -Ras is not explained by an inefficient blockade of Ras activation. Both the ET-R2 cells and the ET-R.X3 cells overexpress the corresponding chimeric EGF/Trk receptors to similar levels (1). Whereas Asn 17 -Ras does not affect the Ca 2ϩ influx following activation of the ET-R receptors which contain the wild-type Trk, the Ca 2ϩ signal observed after stimulation of the ET-RX receptor carrying the mutated PLC binding site is completely abrogated, demonstrating that Asn 17 -Ras is indeed active in the cells expressing the chimeric EGFR/Trk receptors.
The implication of Rac1 in the EGF-induced Ca 2ϩ signal which had been reported by others (23) may indicate an additional requirement for an increased pool of phosphatidylinositol 4,5-bisphosphate. Rac1 and RhoA have been shown to stimulate phosphatidylinositol-4Ј-kinase and phosphatidyl-4phosphate-5Ј-kinase, respectively (40,41). Dominant negative RhoN19 causes indeed a partial inhibition of the EGF-induced Ca 2ϩ signal under conditions where the ATP-mediated Ca 2ϩ transient is unaffected (23). Constitutively active V12Ras has been shown to activate Rac1 (28,29). Although the mechanism by which Ras activates Rac1 is still unclear, evidence is accumulating that Rac1 can be activated by Ras-dependent and independent pathways (29). Evidence for an activation of RhoA by Rac1 has been presented (42). Phospholipase A 2 has also been implicated in the generation of the EGF-induced Ca 2ϩ signal (43). Depending on the cell type, activation of PLA 2 appears to be mediated by either Ras or Rac1, but the role of this PLA 2 -dependent pathway for the EGF-induced Ca 2ϩ signal is still obscure (43).
In summary, activation of the EGFR results in an elevation of cytosolic free Ca 2ϩ which is inhibitable by expression of dominant negative Ras or microinjection of neutralizing anti-Ras antibodies. We conclude that the EGF-induced Ca 2ϩ transient represents a Ras-regulated mechanism and that the rise in cytosolic free Ca 2ϩ elicited by a stimulation of the NGFR is independent of Ras. We suggest, as a hypothetical model, that the Ras dependence of the EGF-induced Ca 2ϩ signal mediated by the EGFR is due to a competition of PLC␥ and p120 Ras-GAP for a common binding site of the EGFR, and that the differences between the EGFR and Trk reflect the differences in the affinity of PLC␥ to the EGFR or Trk, respectively.

FIG. 2. Effect of Asn 17 -Ha-Ras on Ca 2؉ influx induced by the activated exchange mutant receptors ET-RX or EGF-RX.
ET-R.X3 and EGF-R.X2 fibroblasts were transiently transfected with the dominant negative Asn 17 -Ha-Ras construct or the empty control vector and cotransfected with the green fluorescence marker construct by microinjection as described under "Experimental Procedures." 24 h after transfection, the cells were loaded with fura-2, and the Ca 2ϩ influx was estimated from Mn 2ϩ -dependent fura-2 quench detected 60 s after addition of 100 M MnCl 2 (final concentration). Where indicated, the cells were stimulated with EGF and thapsigargin for 1 min. The fura-2 quench was calculated as the percentage of fluorescence decrease of the initial fura-2 fluorescence. The values represent the mean Ϯ S.E. (8 Յ n Յ 24).