Critical Role of Calcium- dependent Epidermal Growth Factor Receptor Transactivation in PC12 Cell Membrane Depolarization and Bradykinin Signaling
1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês
10.1074/jbc.272.40.24767
ISSN1083-351X
AutoresEsther Zwick, Henrik Daub, Naohito Aoki, Yumiko Yamaguchi-Aoki, Inge Tinhofer, Karl Maly, Axel Ullrich,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoPC12 cells respond to a variety of external stimuli such as growth factors, neurotransmitters, and membrane depolarization by activating the Ras/mitogen-activated protein kinase pathway. Here we demonstrate that both depolarization-induced calcium influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Using a tetracycline-controlled expression system in conjunction with a dominant-negative EGFR mutant, we demonstrate that depolarization and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Furthermore, bradykinin-stimulated EGFR transactivation is critically dependent on the presence of extracellular calcium, and when triggered by ionophore treatment, calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, our results establish calcium-dependent EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in neuronal cell types. PC12 cells respond to a variety of external stimuli such as growth factors, neurotransmitters, and membrane depolarization by activating the Ras/mitogen-activated protein kinase pathway. Here we demonstrate that both depolarization-induced calcium influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Using a tetracycline-controlled expression system in conjunction with a dominant-negative EGFR mutant, we demonstrate that depolarization and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Furthermore, bradykinin-stimulated EGFR transactivation is critically dependent on the presence of extracellular calcium, and when triggered by ionophore treatment, calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, our results establish calcium-dependent EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in neuronal cell types. In neurons, the cytosolic calcium concentration is tightly regulated and represents a critical parameter for a variety of intracellular signaling processes. Intracellular calcium levels are modulated either by release of calcium from internal stores or by calcium entry across the plasma membrane through ligand- or voltage-gated calcium channels (1Bootman M.D. Berridge M.J. Cell. 1995; 83: 675-678Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 2Gallin W.J. Greenberg M.E. Curr. Opin. Neurobiol. 1995; 5: 367-374Crossref PubMed Scopus (104) Google Scholar, 3Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1237) Google Scholar). Stimuli such as membrane depolarization result in activation of l-type voltage-sensitive calcium channels, leading to calcium-mediated induction of a specific set of genes and thereby contributing to physiological responses such as neuronal differentiation and survival (4Bading H. Ginty D.D. Greenberg M.E. Science. 1993; 260: 181-186Crossref PubMed Scopus (952) Google Scholar) .Before altering gene expression, elevation of intracellular calcium levels can trigger various signaling events, among them the activation of the small G-protein Ras resulting in stimulation of the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; GPCR, G-protein-coupled receptor; EGF, epidermal growth factor; EGFR, EGF receptor; MBP, myelin basic protein; tTA, tetracycline-controlled transactivator; NGF, nerve growth factor.1The abbreviations used are: MAPK, mitogen-activated protein kinase; GPCR, G-protein-coupled receptor; EGF, epidermal growth factor; EGFR, EGF receptor; MBP, myelin basic protein; tTA, tetracycline-controlled transactivator; NGF, nerve growth factor. pathway (5Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (595) Google Scholar). In PC12 cells, a rat pheochromocytoma cell line widely used as a model system for neuronal differentiation, calcium influx rapidly induces tyrosine phosphorylation of the adaptor protein SHC and SHC-Grb2 complex formation, steps known to couple cell surface receptors such as receptor tyrosine kinases to Ras (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Using a PC12 subline overexpressing a dominant-negative mutant of the cytoplasmatic tyrosine kinase Src, Rusanescu et al. found that inhibiton of membrane depolarization induced SHC tyrosine phosphorylation and MAPK activation (7Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Haleggoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar). Moreover calcium influx following membrane depolarization was recently reported to mediate ligand-independent epidermal growth factor receptor (EGFR) tyrosine phosphorylation in this system (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar). Although direct evidence is lacking regarding whether this represents an essential signaling event for activation of the MAPK pathway, this finding raises the possibility that in PC12 cells calcium may play a role in the EGFR transactivation mechanism as previously demonstrated for signaling through G-protein coupled receptor (GPCR) in Rat-1 fibroblasts (9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar). In addition to membrane depolarization-induced activation of the MAPK pathway (7Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Haleggoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar), GPCR-mediated signaling was also reported to involve Src function (10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, 11Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 12Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Crossref PubMed Scopus (231) Google Scholar, 13Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Moreover, the tyrosine kinase PYK2, a relative of the focal adhesion kinase, was implicated in triggering the MAPK pathway in PC12 cells (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). This raised the possibility that concerted action of receptor tyrosine kinases and cytoplasmatic tyrosine kinases might be necessary to activate certain signaling cascades in response to GPCR stimulation or membrane depolarization (10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar).To analyze potential EGFR function in calcium-dependent signaling, we developed a PC12 cell line that expresses the dominant-negative EGFR mutant HER-CD533 under the control of a tetracycline-sensitive promotor system (14Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4227) Google Scholar). In this system specific EGFR inhibition strongly attenuates SHC tyrosine phosphorylation and MAPK activation in response to both membrane depolarization and bradykinin stimulation. Moreover, we find that calcium ionophore treatment of PC12 cells is sufficient to trigger EGFR tyrosine phosphorylation, whereas EGFR transactivation in response to the GPCR ligand bradykinin is critically dependent on the presence of extracellular calcium. Therefore calcium-dependent EGFR transactivation integrates various extracellular stimuli and provides a link to downstream signal progression.RESULTS AND DISCUSSIONTo analyze EGFR function in response to stimuli such as membrane depolarization or GPCR activation in PC12 cells, we employed the tetracycline-controlled gene expression system for expression of the dominant-negative EGFR mutant HER-CD533 (14Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4227) Google Scholar, 15Redemann N. Holzmann B. von Rüden T. Wagner E.F. Schlessinger J. Ullrich A. Mol. Cell. Biol. 1992; 12: 491-498Crossref PubMed Scopus (119) Google Scholar). This mutant lacks the cytoplasmic domain and disrupts EGFR downstream signaling by forcing endogenous wild-type receptors into signaling-incompetent heterodimers. In the PC12/HER-CD533/Tet cell line, expression of HER-CD533 is suppressed in the presence of tetracycline, whereas removal of tetracycline results in an approximately 20-fold induction within 48 h, as determined by metabolic labeling and subsequent quantification of immunoprecipitated mutant receptor (Fig.1 A). When these cells were subjected to 75 mm extracellular KCl to trigger membrane depolarization in the presence of tetracycline, this treatment stimulated EGFR tyrosine phosphorylation in agreement with previous observations (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar) (Fig. 1 B). The same effect was achieved by treatment of undifferentiated PC12/HER-CD533/Tet cultures with bradykinin in analogy to previously reported findings for the GPCR ligands endothelin-1, lysophosphatidic acid, and thrombin in Rat-1 fibroblasts (9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar). In PC12/HER-CD533/Tet, expression of HER-CD533 upon removal of tetracycline strongly interfered with EGFR tyrosine phosphorylation upon treatment with KCl, bradykinin, and EGF, whereas in control cells these stimuli resulted in increased EGFR tyrosine phosphorylation unaffected by the removal of tetracycline (Fig.1 B). Reprobing with anti-EGFR antibody revealed that comparable amounts of protein were present, which supported the conclusion that EGFR inhibition was due to dominant-negative HER-CD533 action. Importantly, in PC12/HER-CD533/Tet cells elevation of cytoplasmatic calcium upon bradykinin incubation or membrane depolarization was similar when HER-CD533 expression was either induced or repressed (Fig. 1 C), indicating that functional coupling to calcium mobilization was unaffected by EGFR inhibition.Tyrosine phosphorylation of the adaptor protein SHC represents a prominent receptor-proximal signaling step upon EGFR activation (16Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Analysis of crude cell lysates indicated that the HER-CD533 mutant specifically abolished EGF-stimulated SHC tyrosine phosphorylation, whereas the NGF-induced response remained unaltered (Fig.2 A). The identity of the 52-kDa phosphotyrosine-containing protein as the major SHC isoform was confirmed by reprobing immunoblots with a specific antibody. To answer the question of whether the previously reported membrane depolarization- or bradykinin-induced SHC tyrosine phosphorylation (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar) is mediated through transactivation of the EGFR, we immunoprecipitated SHC after stimulation of PC12/HER-CD533/Tet cells in the presence or the absence of tetracyline. As shown in Fig. 2 B, HER-CD533 induction suppressed both KCl- and bradykinin-stimulated SHC tyrosine phosphorylation, demonstrating an essential role of the EGFR in both signals.Figure 2Effect of EGFR inhibition on SHC tyrosine phosphorylation. For expression of HER-CD533, tetracycline was removed as indicated for 48 h. A, quiescent PC12 cells were treated with EGF (1 and 10 ng/ml) or NGF (50 ng/ml) for 90 s and lysed, and crude cell lysates were immunoblotted with αPY antibody (upper panel) following reprobing with polyclonal anti-SHC antibody (lower panel). B, serum-starved cells were treated for 90 s with 75 mm KCl or 1 μm bradykinin and lysed, and SHC was immunoprecipitated (IP) using polyclonal anti-SHC antiserum and immunoblotted with αPY antibody (upper panel), followed by reprobing with anti-SHC antibody (lower panels). Ab, antibody; αPY, anti-phosphotyrosine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next analyzed the role of EGFR function in the activation of the MAPK pathway in PC12 cells. To address this question, we examined the effect of EGFR inhibition on activation of ERK-2 following KCl or bradykinin treatment. ERK-2 activity was measured with an immunocomplex kinase assay using myelin basic protein as an exogenous substrate. As shown in Fig. 3 (upper panel), expression of HER-CD533 strongly and reproducibly attenuated ERK-2 activation upon KCl or bradykinin treatment by approximately 80%. For reasons currently unknown, the stimulation of MAPK activity following bradykinin treatment was reproducibly weaker compared with stimulation with KCl. As expected, EGF-induced MAPK stimulation was completely suppressed, whereas the NGF-induced response, included as a control, was not significantly affected. Interestingly, despite weaker stimulation of Shc tyrosine phosphorylation (Fig. 2 A), NGF induced MAPK activity as potently as 1 ng/ml EGF. This suggested that upon NGF stimulation additional signal transducers such as the recently described Grb2-binding protein FRS2 may contribute to the activation of the Ras/MAPK pathway (17Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar).Figure 3Effect of EGFR inhibition on stimulation of MAPK activity. PC12/HER-CD533/Tet (upper panel) and PC12/control cells (lower panel) were pretreated as indicated with or without tetracyline for 48 h and serum-starved for 20 h. Cells were stimulated for 5 min with 75 mmKCl, 1 μm bradykinin, 1 ng/ml EGF or NGF and lysed, and endogenous ERK2 activity was determined using MBP as described under "Experimental Procedures." Phosphorylated MBP was visualized by autoradiography after gel electrophoresis. IP, immunoprecipitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, any influence of tetracycline on MAPK activation was excluded using control cells (Fig. 3, lower panel). Similar effects on MAPK activation upon these treatments were obtained with nanomolar concentrations of the EGFR-specific inhibitor AG1478 (data not shown).The recently reported finding of ligand-independent EGFR tyrosine phosphorylation upon membrane depolarization-mediated calcium influx (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar) raised the question of whether calcium may be critical for EGFR transactivation in PC12 cells. As shown in Fig.4 A, elimination of extracellular calcium with 3 mm EGTA for 5 min completely abolished the increase of EGFR tyrosine phosphorylation upon bradykinin stimulation. Moreover, when we used the calcium ionophore ionomycin to directly elevate intracellular calcium levels, enhanced phosphorylation of the EGFR on tyrosine was readily detected. Tetracycline controlled expression of HER-CD533 strongly interfered with ionomycin-induced EGFR tyrosine phosphorylation (Fig. 4 B), demonstrating that calcium is both necessary and sufficient for ligand-independent EGFR transactivation in PC12 cells. Interestingly EGFR inhibition differentially reduced ERK2 activation levels induced by treatment with ionomycin or KCl and bradykinin by 50 and 80%, respectively (Figs. 3and 4 C). This quantitative difference could be due to a differential parallel involvement of the calcium-dependent Ras-GRF in the activation of a tyrosine kinase-independent Ras/MAPK pathway in neuronal cells (18Buchsbaum R. Telliez J.B. Goonesekera S. Feig L.A. Mol. Cell. Biol. 1996; 16: 4888-4896Crossref PubMed Scopus (91) Google Scholar).Figure 4Influence of calcium on EGFR transactivation in PC12 cells. A, quiescent PC12 cells were pretreated as indicated with 3 mm EGTA for 5 min prior to stimulation with 1 μm bradykinin. After cell lysis, EGFR was immunoprecipitated (IP) with rabbit polyclonal anti-EGFR antibody (Ab). Tyrosine-phosphorylated EGFR was detected by immunoblotting with αPY antibody (upper panel) followed by reprobing with anti-EGFR antibody (lower panel).B, serum-starved PC12/HER-CD533/Tet cells were pretreated with or without tetracycline for 48 h, stimulated with vehicle or with 1.8 μm ionomycin for the indicated time, and lysed. Tyrosine-phosphorylated EGFR was detected as described above.C, quiescent PC12 cells were stimulated with 1.8 μm ionomycin and lysed, and endogenous ERK2 activity was determined using MBP as described under "Experimental Procedures." Phosphorylated MBP was visualized by autoradiography after gel electrophoresis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly, elevation of intracellular calcium levels was also reported to trigger tyrosine phosphorylation and activation of the cytoplasmatic tyrosine kinase PYK2 in PC12 cells (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Moreover, this tyrosine kinase had been suggested to link stimuli such as membrane depolarization and bradykinin to MAPK activation. Because PYK2 was reported to interact with Src upon bradykinin stimulation (10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar), this association might be required for Src to efficiently phosphorylate its cellular substrates on tyrosine residues. Moreover, because overexpression of a dominant-negative Src mutant had been reported to interfere with depolarization-induced SHC tyrosine phosphorylation (7Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Haleggoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar), our findings raise the question how the EGFR and the cytoplasmatic tyrosine kinase complex Src/PYK2 are functionally linked in PC12 cells. Because oncogenic Src appears to activate the EGFR by tyrosine phosphorylation at nonregular sites (19Wasilenko W.J. Payne D.M. Fitzgerald D.L. Weber M.J. Mol. Cell. Biol. 1991; 11: 309-321Crossref PubMed Scopus (63) Google Scholar), Src and PYK2 could act upstream and utilize EGFR for further signal transmission. Alternatively, to reconcile the results presented here and those reported earlier (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar, 10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar), EGFR transactivation might occur independently and parallel to Src/PYK2 with SHC binding to phosphorylated EGFR and subsequent SHC phosphorylation by activated Src.The tetracycline-controlled inducible expression system used in this study to analyze EGFR function in PC12 cells may be instrumental for finding answers to these questions. Because, at the concentrations used, tetracycline is without any detectable effect and the temporal and quantitative parameters of mutant EGFR expression can be tightly controlled, this system provides an excellent tool for the examination of multiple elements in the cellular signal transmission network of the PC12 model system. In the context of the nervous system where the EGFR is found in various areas (20Tucker M.S. Khan I. Fuchs-Young R. Price S. Steininger T.L. Greene G. Wainer B.H. Rosner M.R. Brain Res. 1993; 631: 65-71Crossref PubMed Scopus (52) Google Scholar), our findings provide a new basis for the investigation of GPCR mediated signals and their significance for biological phenomena such as neuronal cell survival and neurodegenerative disease. In neurons, the cytosolic calcium concentration is tightly regulated and represents a critical parameter for a variety of intracellular signaling processes. Intracellular calcium levels are modulated either by release of calcium from internal stores or by calcium entry across the plasma membrane through ligand- or voltage-gated calcium channels (1Bootman M.D. Berridge M.J. Cell. 1995; 83: 675-678Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 2Gallin W.J. Greenberg M.E. Curr. Opin. Neurobiol. 1995; 5: 367-374Crossref PubMed Scopus (104) Google Scholar, 3Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1237) Google Scholar). Stimuli such as membrane depolarization result in activation of l-type voltage-sensitive calcium channels, leading to calcium-mediated induction of a specific set of genes and thereby contributing to physiological responses such as neuronal differentiation and survival (4Bading H. Ginty D.D. Greenberg M.E. Science. 1993; 260: 181-186Crossref PubMed Scopus (952) Google Scholar) . Before altering gene expression, elevation of intracellular calcium levels can trigger various signaling events, among them the activation of the small G-protein Ras resulting in stimulation of the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; GPCR, G-protein-coupled receptor; EGF, epidermal growth factor; EGFR, EGF receptor; MBP, myelin basic protein; tTA, tetracycline-controlled transactivator; NGF, nerve growth factor.1The abbreviations used are: MAPK, mitogen-activated protein kinase; GPCR, G-protein-coupled receptor; EGF, epidermal growth factor; EGFR, EGF receptor; MBP, myelin basic protein; tTA, tetracycline-controlled transactivator; NGF, nerve growth factor. pathway (5Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (595) Google Scholar). In PC12 cells, a rat pheochromocytoma cell line widely used as a model system for neuronal differentiation, calcium influx rapidly induces tyrosine phosphorylation of the adaptor protein SHC and SHC-Grb2 complex formation, steps known to couple cell surface receptors such as receptor tyrosine kinases to Ras (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Using a PC12 subline overexpressing a dominant-negative mutant of the cytoplasmatic tyrosine kinase Src, Rusanescu et al. found that inhibiton of membrane depolarization induced SHC tyrosine phosphorylation and MAPK activation (7Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Haleggoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar). Moreover calcium influx following membrane depolarization was recently reported to mediate ligand-independent epidermal growth factor receptor (EGFR) tyrosine phosphorylation in this system (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar). Although direct evidence is lacking regarding whether this represents an essential signaling event for activation of the MAPK pathway, this finding raises the possibility that in PC12 cells calcium may play a role in the EGFR transactivation mechanism as previously demonstrated for signaling through G-protein coupled receptor (GPCR) in Rat-1 fibroblasts (9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar). In addition to membrane depolarization-induced activation of the MAPK pathway (7Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Haleggoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar), GPCR-mediated signaling was also reported to involve Src function (10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, 11Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 12Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Crossref PubMed Scopus (231) Google Scholar, 13Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Moreover, the tyrosine kinase PYK2, a relative of the focal adhesion kinase, was implicated in triggering the MAPK pathway in PC12 cells (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). This raised the possibility that concerted action of receptor tyrosine kinases and cytoplasmatic tyrosine kinases might be necessary to activate certain signaling cascades in response to GPCR stimulation or membrane depolarization (10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar). To analyze potential EGFR function in calcium-dependent signaling, we developed a PC12 cell line that expresses the dominant-negative EGFR mutant HER-CD533 under the control of a tetracycline-sensitive promotor system (14Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4227) Google Scholar). In this system specific EGFR inhibition strongly attenuates SHC tyrosine phosphorylation and MAPK activation in response to both membrane depolarization and bradykinin stimulation. Moreover, we find that calcium ionophore treatment of PC12 cells is sufficient to trigger EGFR tyrosine phosphorylation, whereas EGFR transactivation in response to the GPCR ligand bradykinin is critically dependent on the presence of extracellular calcium. Therefore calcium-dependent EGFR transactivation integrates various extracellular stimuli and provides a link to downstream signal progression. RESULTS AND DISCUSSIONTo analyze EGFR function in response to stimuli such as membrane depolarization or GPCR activation in PC12 cells, we employed the tetracycline-controlled gene expression system for expression of the dominant-negative EGFR mutant HER-CD533 (14Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4227) Google Scholar, 15Redemann N. Holzmann B. von Rüden T. Wagner E.F. Schlessinger J. Ullrich A. Mol. Cell. Biol. 1992; 12: 491-498Crossref PubMed Scopus (119) Google Scholar). This mutant lacks the cytoplasmic domain and disrupts EGFR downstream signaling by forcing endogenous wild-type receptors into signaling-incompetent heterodimers. In the PC12/HER-CD533/Tet cell line, expression of HER-CD533 is suppressed in the presence of tetracycline, whereas removal of tetracycline results in an approximately 20-fold induction within 48 h, as determined by metabolic labeling and subsequent quantification of immunoprecipitated mutant receptor (Fig.1 A). When these cells were subjected to 75 mm extracellular KCl to trigger membrane depolarization in the presence of tetracycline, this treatment stimulated EGFR tyrosine phosphorylation in agreement with previous observations (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar) (Fig. 1 B). The same effect was achieved by treatment of undifferentiated PC12/HER-CD533/Tet cultures with bradykinin in analogy to previously reported findings for the GPCR ligands endothelin-1, lysophosphatidic acid, and thrombin in Rat-1 fibroblasts (9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar). In PC12/HER-CD533/Tet, expression of HER-CD533 upon removal of tetracycline strongly interfered with EGFR tyrosine phosphorylation upon treatment with KCl, bradykinin, and EGF, whereas in control cells these stimuli resulted in increased EGFR tyrosine phosphorylation unaffected by the removal of tetracycline (Fig.1 B). Reprobing with anti-EGFR antibody revealed that comparable amounts of protein were present, which supported the conclusion that EGFR inhibition was due to dominant-negative HER-CD533 action. Importantly, in PC12/HER-CD533/Tet cells elevation of cytoplasmatic calcium upon bradykinin incubation or membrane depolarization was similar when HER-CD533 expression was either induced or repressed (Fig. 1 C), indicating that functional coupling to calcium mobilization was unaffected by EGFR inhibition.Tyrosine phosphorylation of the adaptor protein SHC represents a prominent receptor-proximal signaling step upon EGFR activation (16Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Analysis of crude cell lysates indicated that the HER-CD533 mutant specifically abolished EGF-stimulated SHC tyrosine phosphorylation, whereas the NGF-induced response remained unaltered (Fig.2 A). The identity of the 52-kDa phosphotyrosine-containing protein as the major SHC isoform was confirmed by reprobing immunoblots with a specific antibody. To answer the question of whether the previously reported membrane depolarization- or bradykinin-induced SHC tyrosine phosphorylation (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar) is mediated through transactivation of the EGFR, we immunoprecipitated SHC after stimulation of PC12/HER-CD533/Tet cells in the presence or the absence of tetracyline. As shown in Fig. 2 B, HER-CD533 induction suppressed both KCl- and bradykinin-stimulated SHC tyrosine phosphorylation, demonstrating an essential role of the EGFR in both signals.We next analyzed the role of EGFR function in the activation of the MAPK pathway in PC12 cells. To address this question, we examined the effect of EGFR inhibition on activation of ERK-2 following KCl or bradykinin treatment. ERK-2 activity was measured with an immunocomplex kinase assay using myelin basic protein as an exogenous substrate. As shown in Fig. 3 (upper panel), expression of HER-CD533 strongly and reproducibly attenuated ERK-2 activation upon KCl or bradykinin treatment by approximately 80%. For reasons currently unknown, the stimulation of MAPK activity following bradykinin treatment was reproducibly weaker compared with stimulation with KCl. As expected, EGF-induced MAPK stimulation was completely suppressed, whereas the NGF-induced response, included as a control, was not significantly affected. Interestingly, despite weaker stimulation of Shc tyrosine phosphorylation (Fig. 2 A), NGF induced MAPK activity as potently as 1 ng/ml EGF. This suggested that upon NGF stimulation additional signal transducers such as the recently described Grb2-binding protein FRS2 may contribute to the activation of the Ras/MAPK pathway (17Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar).Figure 3Effect of EGFR inhibition on stimulation of MAPK activity. PC12/HER-CD533/Tet (upper panel) and PC12/control cells (lower panel) were pretreated as indicated with or without tetracyline for 48 h and serum-starved for 20 h. Cells were stimulated for 5 min with 75 mmKCl, 1 μm bradykinin, 1 ng/ml EGF or NGF and lysed, and endogenous ERK2 activity was determined using MBP as described under "Experimental Procedures." Phosphorylated MBP was visualized by autoradiography after gel electrophoresis. IP, immunoprecipitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, any influence of tetracycline on MAPK activation was excluded using control cells (Fig. 3, lower panel). Similar effects on MAPK activation upon these treatments were obtained with nanomolar concentrations of the EGFR-specific inhibitor AG1478 (data not shown).The recently reported finding of ligand-independent EGFR tyrosine phosphorylation upon membrane depolarization-mediated calcium influx (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar) raised the question of whether calcium may be critical for EGFR transactivation in PC12 cells. As shown in Fig.4 A, elimination of extracellular calcium with 3 mm EGTA for 5 min completely abolished the increase of EGFR tyrosine phosphorylation upon bradykinin stimulation. Moreover, when we used the calcium ionophore ionomycin to directly elevate intracellular calcium levels, enhanced phosphorylation of the EGFR on tyrosine was readily detected. Tetracycline controlled expression of HER-CD533 strongly interfered with ionomycin-induced EGFR tyrosine phosphorylation (Fig. 4 B), demonstrating that calcium is both necessary and sufficient for ligand-independent EGFR transactivation in PC12 cells. Interestingly EGFR inhibition differentially reduced ERK2 activation levels induced by treatment with ionomycin or KCl and bradykinin by 50 and 80%, respectively (Figs. 3and 4 C). This quantitative difference could be due to a differential parallel involvement of the calcium-dependent Ras-GRF in the activation of a tyrosine kinase-independent Ras/MAPK pathway in neuronal cells (18Buchsbaum R. Telliez J.B. Goonesekera S. Feig L.A. Mol. Cell. Biol. 1996; 16: 4888-4896Crossref PubMed Scopus (91) Google Scholar).Figure 4Influence of calcium on EGFR transactivation in PC12 cells. A, quiescent PC12 cells were pretreated as indicated with 3 mm EGTA for 5 min prior to stimulation with 1 μm bradykinin. After cell lysis, EGFR was immunoprecipitated (IP) with rabbit polyclonal anti-EGFR antibody (Ab). Tyrosine-phosphorylated EGFR was detected by immunoblotting with αPY antibody (upper panel) followed by reprobing with anti-EGFR antibody (lower panel).B, serum-starved PC12/HER-CD533/Tet cells were pretreated with or without tetracycline for 48 h, stimulated with vehicle or with 1.8 μm ionomycin for the indicated time, and lysed. Tyrosine-phosphorylated EGFR was detected as described above.C, quiescent PC12 cells were stimulated with 1.8 μm ionomycin and lysed, and endogenous ERK2 activity was determined using MBP as described under "Experimental Procedures." Phosphorylated MBP was visualized by autoradiography after gel electrophoresis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly, elevation of intracellular calcium levels was also reported to trigger tyrosine phosphorylation and activation of the cytoplasmatic tyrosine kinase PYK2 in PC12 cells (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Moreover, this tyrosine kinase had been suggested to link stimuli such as membrane depolarization and bradykinin to MAPK activation. Because PYK2 was reported to interact with Src upon bradykinin stimulation (10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar), this association might be required for Src to efficiently phosphorylate its cellular substrates on tyrosine residues. Moreover, because overexpression of a dominant-negative Src mutant had been reported to interfere with depolarization-induced SHC tyrosine phosphorylation (7Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Haleggoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar), our findings raise the question how the EGFR and the cytoplasmatic tyrosine kinase complex Src/PYK2 are functionally linked in PC12 cells. Because oncogenic Src appears to activate the EGFR by tyrosine phosphorylation at nonregular sites (19Wasilenko W.J. Payne D.M. Fitzgerald D.L. Weber M.J. Mol. Cell. Biol. 1991; 11: 309-321Crossref PubMed Scopus (63) Google Scholar), Src and PYK2 could act upstream and utilize EGFR for further signal transmission. Alternatively, to reconcile the results presented here and those reported earlier (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar, 10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar), EGFR transactivation might occur independently and parallel to Src/PYK2 with SHC binding to phosphorylated EGFR and subsequent SHC phosphorylation by activated Src.The tetracycline-controlled inducible expression system used in this study to analyze EGFR function in PC12 cells may be instrumental for finding answers to these questions. Because, at the concentrations used, tetracycline is without any detectable effect and the temporal and quantitative parameters of mutant EGFR expression can be tightly controlled, this system provides an excellent tool for the examination of multiple elements in the cellular signal transmission network of the PC12 model system. In the context of the nervous system where the EGFR is found in various areas (20Tucker M.S. Khan I. Fuchs-Young R. Price S. Steininger T.L. Greene G. Wainer B.H. Rosner M.R. Brain Res. 1993; 631: 65-71Crossref PubMed Scopus (52) Google Scholar), our findings provide a new basis for the investigation of GPCR mediated signals and their significance for biological phenomena such as neuronal cell survival and neurodegenerative disease. To analyze EGFR function in response to stimuli such as membrane depolarization or GPCR activation in PC12 cells, we employed the tetracycline-controlled gene expression system for expression of the dominant-negative EGFR mutant HER-CD533 (14Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4227) Google Scholar, 15Redemann N. Holzmann B. von Rüden T. Wagner E.F. Schlessinger J. Ullrich A. Mol. Cell. Biol. 1992; 12: 491-498Crossref PubMed Scopus (119) Google Scholar). This mutant lacks the cytoplasmic domain and disrupts EGFR downstream signaling by forcing endogenous wild-type receptors into signaling-incompetent heterodimers. In the PC12/HER-CD533/Tet cell line, expression of HER-CD533 is suppressed in the presence of tetracycline, whereas removal of tetracycline results in an approximately 20-fold induction within 48 h, as determined by metabolic labeling and subsequent quantification of immunoprecipitated mutant receptor (Fig.1 A). When these cells were subjected to 75 mm extracellular KCl to trigger membrane depolarization in the presence of tetracycline, this treatment stimulated EGFR tyrosine phosphorylation in agreement with previous observations (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar) (Fig. 1 B). The same effect was achieved by treatment of undifferentiated PC12/HER-CD533/Tet cultures with bradykinin in analogy to previously reported findings for the GPCR ligands endothelin-1, lysophosphatidic acid, and thrombin in Rat-1 fibroblasts (9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar). In PC12/HER-CD533/Tet, expression of HER-CD533 upon removal of tetracycline strongly interfered with EGFR tyrosine phosphorylation upon treatment with KCl, bradykinin, and EGF, whereas in control cells these stimuli resulted in increased EGFR tyrosine phosphorylation unaffected by the removal of tetracycline (Fig.1 B). Reprobing with anti-EGFR antibody revealed that comparable amounts of protein were present, which supported the conclusion that EGFR inhibition was due to dominant-negative HER-CD533 action. Importantly, in PC12/HER-CD533/Tet cells elevation of cytoplasmatic calcium upon bradykinin incubation or membrane depolarization was similar when HER-CD533 expression was either induced or repressed (Fig. 1 C), indicating that functional coupling to calcium mobilization was unaffected by EGFR inhibition. Tyrosine phosphorylation of the adaptor protein SHC represents a prominent receptor-proximal signaling step upon EGFR activation (16Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Analysis of crude cell lysates indicated that the HER-CD533 mutant specifically abolished EGF-stimulated SHC tyrosine phosphorylation, whereas the NGF-induced response remained unaltered (Fig.2 A). The identity of the 52-kDa phosphotyrosine-containing protein as the major SHC isoform was confirmed by reprobing immunoblots with a specific antibody. To answer the question of whether the previously reported membrane depolarization- or bradykinin-induced SHC tyrosine phosphorylation (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar) is mediated through transactivation of the EGFR, we immunoprecipitated SHC after stimulation of PC12/HER-CD533/Tet cells in the presence or the absence of tetracyline. As shown in Fig. 2 B, HER-CD533 induction suppressed both KCl- and bradykinin-stimulated SHC tyrosine phosphorylation, demonstrating an essential role of the EGFR in both signals. We next analyzed the role of EGFR function in the activation of the MAPK pathway in PC12 cells. To address this question, we examined the effect of EGFR inhibition on activation of ERK-2 following KCl or bradykinin treatment. ERK-2 activity was measured with an immunocomplex kinase assay using myelin basic protein as an exogenous substrate. As shown in Fig. 3 (upper panel), expression of HER-CD533 strongly and reproducibly attenuated ERK-2 activation upon KCl or bradykinin treatment by approximately 80%. For reasons currently unknown, the stimulation of MAPK activity following bradykinin treatment was reproducibly weaker compared with stimulation with KCl. As expected, EGF-induced MAPK stimulation was completely suppressed, whereas the NGF-induced response, included as a control, was not significantly affected. Interestingly, despite weaker stimulation of Shc tyrosine phosphorylation (Fig. 2 A), NGF induced MAPK activity as potently as 1 ng/ml EGF. This suggested that upon NGF stimulation additional signal transducers such as the recently described Grb2-binding protein FRS2 may contribute to the activation of the Ras/MAPK pathway (17Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar). Furthermore, any influence of tetracycline on MAPK activation was excluded using control cells (Fig. 3, lower panel). Similar effects on MAPK activation upon these treatments were obtained with nanomolar concentrations of the EGFR-specific inhibitor AG1478 (data not shown). The recently reported finding of ligand-independent EGFR tyrosine phosphorylation upon membrane depolarization-mediated calcium influx (8Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar) raised the question of whether calcium may be critical for EGFR transactivation in PC12 cells. As shown in Fig.4 A, elimination of extracellular calcium with 3 mm EGTA for 5 min completely abolished the increase of EGFR tyrosine phosphorylation upon bradykinin stimulation. Moreover, when we used the calcium ionophore ionomycin to directly elevate intracellular calcium levels, enhanced phosphorylation of the EGFR on tyrosine was readily detected. Tetracycline controlled expression of HER-CD533 strongly interfered with ionomycin-induced EGFR tyrosine phosphorylation (Fig. 4 B), demonstrating that calcium is both necessary and sufficient for ligand-independent EGFR transactivation in PC12 cells. Interestingly EGFR inhibition differentially reduced ERK2 activation levels induced by treatment with ionomycin or KCl and bradykinin by 50 and 80%, respectively (Figs. 3and 4 C). This quantitative difference could be due to a differential parallel involvement of the calcium-dependent Ras-GRF in the activation of a tyrosine kinase-independent Ras/MAPK pathway in neuronal cells (18Buchsbaum R. Telliez J.B. Goonesekera S. Feig L.A. Mol. Cell. Biol. 1996; 16: 4888-4896Crossref PubMed Scopus (91) Google Scholar). Interestingly, elevation of intracellular calcium levels was also reported to trigger tyrosine phosphorylation and activation of the cytoplasmatic tyrosine kinase PYK2 in PC12 cells (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Moreover, this tyrosine kinase had been suggested to link stimuli such as membrane depolarization and bradykinin to MAPK activation. Because PYK2 was reported to interact with Src upon bradykinin stimulation (10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar), this association might be required for Src to efficiently phosphorylate its cellular substrates on tyrosine residues. Moreover, because overexpression of a dominant-negative Src mutant had been reported to interfere with depolarization-induced SHC tyrosine phosphorylation (7Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Haleggoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar), our findings raise the question how the EGFR and the cytoplasmatic tyrosine kinase complex Src/PYK2 are functionally linked in PC12 cells. Because oncogenic Src appears to activate the EGFR by tyrosine phosphorylation at nonregular sites (19Wasilenko W.J. Payne D.M. Fitzgerald D.L. Weber M.J. Mol. Cell. Biol. 1991; 11: 309-321Crossref PubMed Scopus (63) Google Scholar), Src and PYK2 could act upstream and utilize EGFR for further signal transmission. Alternatively, to reconcile the results presented here and those reported earlier (6Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar, 10Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar), EGFR transactivation might occur independently and parallel to Src/PYK2 with SHC binding to phosphorylated EGFR and subsequent SHC phosphorylation by activated Src. The tetracycline-controlled inducible expression system used in this study to analyze EGFR function in PC12 cells may be instrumental for finding answers to these questions. Because, at the concentrations used, tetracycline is without any detectable effect and the temporal and quantitative parameters of mutant EGFR expression can be tightly controlled, this system provides an excellent tool for the examination of multiple elements in the cellular signal transmission network of the PC12 model system. In the context of the nervous system where the EGFR is found in various areas (20Tucker M.S. Khan I. Fuchs-Young R. Price S. Steininger T.L. Greene G. Wainer B.H. Rosner M.R. Brain Res. 1993; 631: 65-71Crossref PubMed Scopus (52) Google Scholar), our findings provide a new basis for the investigation of GPCR mediated signals and their significance for biological phenomena such as neuronal cell survival and neurodegenerative disease. We thank Dr. T. Haller for technical help with the fluorescence measurements, Drs. H. Bujard and M. Gossen for providing us the plasmids pUHD15-1neo and pUHD10-3, and Dr. C. Wallasch for helpful discussions.
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