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• W2022321394 abstract "The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1–7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr402. This was reduced by Ca2+ chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKCα and -ε to the cell membrane and enhanced the association of Src with PKCα and PKCε, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant–negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons. The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1–7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr402. This was reduced by Ca2+ chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKCα and -ε to the cell membrane and enhanced the association of Src with PKCα and PKCε, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant–negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons. gonadotropin-releasing hormone C-terminal Src kinase epidermal growth factor epidermal growth factor receptor extracellularly regulated MAPKs 1 and 2 phosphorylated ERK1/2 G protein-coupled receptor phorbol 12-myristate 13-acetate proline-rich tyrosine kinase dominant-negative Pyk2 mutant receptor tyrosine kinase protein kinase C focal adhesion kinase phosphate-buffered saline basic fibroblast growth factor heparin-binding EGF 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine 1,2-bis (2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid The hypothalamic decapeptide, gonadotropin releasing hormone (GnRH),1 is a primary regulatory factor in the neuroendocrine control of reproduction and is released in an episodic manner from the hypothalamic GnRH neurons. The pulsatile delivery of GnRH to the anterior pituitary gland is essential to maintain the circulating gonadotropin profiles that are necessary for normal reproductive function. In addition to regulating pituitary gonadotropin release, GnRH has extrapituitary actions in neural and nonneural tissues and in several types of tumor cells (1Stojilkovic S.S. Catt K.J. J. Neuroendocrinol. 1995; 7: 739-757Google Scholar). Immortalized GnRH-producing neurons (GT1–7 neurons) express several G protein-coupled receptors (GPCRs), including those for GnRH and luteinizing hormone/human chorionic gonadotropin (2Mores N. Krsmanovic L.Z. Catt K.J. Endocrinology. 1996; 137: 5731-5734Google Scholar, 3Krsmanovic L.Z. Stojilkovic S.S. Mertz L.M. Tomic M. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3908-3912Google Scholar), as well as α- and β-adrenergic (4Martinez de la Escalera G. Choi A.L. Weiner R.I. Endocrinology. 1992; 131: 1397-1402Google Scholar), muscarinic (5Krsmanovic L.Z. Mores N. Navarro C.E. Saeed S.A. Arora K.K. Catt K.J. Endocrinology. 1998; 139: 4037-4043Google Scholar), and serotonergic receptors (6Hery M. Francois-Bellan A.M. Hery F. Deprez P. Becquet D. Endocrine. 1997; 7: 261-265Google Scholar). These cells retain many of the characteristics of the native GnRH neurons, including the ability to maintain pulsatile GnRH release (1Stojilkovic S.S. Catt K.J. J. Neuroendocrinol. 1995; 7: 739-757Google Scholar,3Krsmanovic L.Z. Stojilkovic S.S. Mertz L.M. Tomic M. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3908-3912Google Scholar). Recent evidence suggests that the autocrine action of GnRH on hypothalamic GnRH neurons is involved in the mechanism of pulsatile GnRH secretion (3Krsmanovic L.Z. Stojilkovic S.S. Mertz L.M. Tomic M. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3908-3912Google Scholar). Agonist activation of specific GPCRs and the resulting dissociation of their cognate G proteins releases α- and βγ-subunits that regulate phospholipase C-β, adenylyl cyclase, and ion channels, which in turn control the intracellular levels of inositol phosphates, Ca2+, cAMP, and other second messengers (7Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Google Scholar, 8Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Google Scholar). The major signal transduction pathways in cells expressing GnRH receptors are initiated by activation of phospholipase C. The consequent calcium (Ca2+) mobilization and activation of protein kinase C (PKC) by GnRH are key elements in the hypothalamic control of gonadotropin secretion from the anterior pituitary gland (1Stojilkovic S.S. Catt K.J. J. Neuroendocrinol. 1995; 7: 739-757Google Scholar, 3Krsmanovic L.Z. Stojilkovic S.S. Mertz L.M. Tomic M. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3908-3912Google Scholar, 5Krsmanovic L.Z. Mores N. Navarro C.E. Saeed S.A. Arora K.K. Catt K.J. Endocrinology. 1998; 139: 4037-4043Google Scholar). Activation of PKC and Ca2+ mobilization during GnRH receptor stimulation are also responsible for mediating downstream signals leading to activation of extracellularly regulated mitogen-activated protein kinases (ERK1/2 MAPKs) that transmit signals from the cell surface to the nucleus to regulate transcriptional and other processes (7Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Google Scholar, 8Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Google Scholar, 9Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Google Scholar, 10Grosse R. Roelle S. Herrlich A. Hohn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Google Scholar, 11Han X-B. Conn P.M. Endocrinology. 1999; 140: 2241-2251Google Scholar, 12Mulvaney J.M. Zhang T. Fewtrell C. Roberson M.S. J. Biol. Chem. 1999; 274: 29796-29804Google Scholar, 13Reiss N. Levi L.N. Shachm S. Harris D. Seger R. Naor Z. Endocrinology. 1997; 138: 1673-1682Google Scholar). However, the specific PKC isoforms that are involved in GnRH-induced ERK1/2 activation in GT1–7 cells are not known. Mitogenic signaling by GPCRs can also occur through activation of tyrosine kinases of the Src family, focal adhesion kinases (FAKs), and receptor tyrosine kinases (RTKs). The RTKs involved in GPCR-mediated activation of ERK1/2 MAPKs include the EGF-R, platelet-derived growth factor receptor, and insulin-like growth factor receptor (14Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Google Scholar, 15Mondorf U.F. Geiger H. Herrero M. Zeuzem S. Piiper A. FEBS Lett. 2000; 472: 129-132Google Scholar, 16Roudabush F.L. Pierce K.L. Maudsley S. Khan K.D. Luttrell L.M. J. Biol. Chem. 2000; 275: 22583-222589Google Scholar). The GPCRs mediating EGF-R transactivation during agonist stimulation include the AT1 angiotensin receptor (17Saito Y. Berk B.C. J. Mol. Cell Cardiol. 2001; 33: 3-7Google Scholar), the β-adrenoreceptor (18Maudsley S. Pierce K.L. Zamah A.M. Miller W.E. Ahn S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2000; 275: 9572-9580Google Scholar), the P2Y2 purinoceptor (19Soltoff S.P. J. Biol. Chem. 1998; 273: 23110-23117Google Scholar), and receptors for endothelin-1, thrombin, lysophosphatidic acid, and bradykinin (20Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Google Scholar, 21Della Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: 13978-13984Google Scholar). GPCR-mediated transactivation of the EGF-R initiates the ERK1/2 MAPK cascade through recruitment of adaptor proteins, such as the Shc-Grb2-Sos complex, that activate the small G protein, Ras (14Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Google Scholar, 22Gschwind A. Zwick E. Prenzel N. Leserer M. Ullrich A. Oncogene. 2001; 20: 1594-1600Google Scholar). Depending on the GPCR agonist and cell type, Ca2+, PKC, G protein βγ subunits, and nonreceptor tyrosine kinases including Src and Pyk2, have been implicated in GPCR-induced EGF-R transactivation (14Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Google Scholar, 22Gschwind A. Zwick E. Prenzel N. Leserer M. Ullrich A. Oncogene. 2001; 20: 1594-1600Google Scholar). Endogenous EGF-Rs are expressed in several model systems, including αT3–1 gonadotrophs, COS-7 cells, and HEK-293 cells, that have been used in studies on GnRH signaling. However, the role of EGF-R transactivation in GnRH-induced ERK activation has been a subject of controversy and is not clearly defined (9Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Google Scholar, 10Grosse R. Roelle S. Herrlich A. Hohn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Google Scholar, 23Hislop J.N. Everest H.M. Flynn A. Harding T. Uney J.B. Troskie B.E. Millar R.P. McArdle C.A. J. Biol. Chem. 2001; 276: 39685-39694Google Scholar). Also, the signaling molecules involved in cross-talk between the neuronal GnRH-R and the EGF-R have not been identified. Depending upon the cell type, GPCRs mediate both Ras-independent ERK1/2 activation via stimulation of PKC and Ras-dependent ERK activation by receptor and nonreceptor tyrosine kinases (7Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Google Scholar, 14Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Google Scholar). GnRH has been found to activate ERK1/2 MAPKs in αT3–1 gonadotrophs and in COS-7 cells (8Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Google Scholar, 9Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Google Scholar, 10Grosse R. Roelle S. Herrlich A. Hohn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Google Scholar, 12Mulvaney J.M. Zhang T. Fewtrell C. Roberson M.S. J. Biol. Chem. 1999; 274: 29796-29804Google Scholar, 13Reiss N. Levi L.N. Shachm S. Harris D. Seger R. Naor Z. Endocrinology. 1997; 138: 1673-1682Google Scholar) and GH3 cells transfected with the GnRH receptor (11Han X-B. Conn P.M. Endocrinology. 1999; 140: 2241-2251Google Scholar). It also stimulates Jun N-terminal kinase in αT3–1 cells (24Levi N. Hanoch T. Benard O. Rozenblat M. Harris D. Reiss N. Naor Z. Seger R. Mol. Endocrinol. 1998; 12: 815-824Google Scholar) and p38-MAPK in LβT2 gonadotrophs (25Liu F. Austin D.A. Mellon P.L. Olefsky J.M. Webster N.J.G. Mol. Endocrinol. 2002; 16: 419-434Google Scholar). Activation of these MAPKs by other GPCRs, such as angiotensin II (26Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owasa K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Google Scholar, 27Matsubara H. Shibasaki Y. Okigaki M. Mori Y. Masaki H. Kosaki A. Tsutsumi Y. Biochem. Biophys. Res. Commun. 2001; 282: 1085-10891Google Scholar), endothelin (28Sorokin A. Kozlowski P. Graves L. Philip A. J. Biol. Chem. 2001; 276: 21521-21528Google Scholar), adrenomedullin (29Iwasaki H. Shichiri M. Marumo F. Hirata Y. Endocrinology. 2001; 142: 564-572Google Scholar), and acetylcholine (30Keely S.J. Calandrella S.O. Barrett K.E. J. Biol. Chem. 2000; 275: 12619-12625Google Scholar), is mediated through the proline-rich protein tyrosine kinase, Pyk2. In general, Pyk2 activation in conjunction with Src kinase appears to be a key element in GPCR-mediated transactivation of the EGF-R (31Andreev J. Galisteo M.L. Kranenburg O. Logan S.K. Chiu E.S. Okigaki M. Cary L.A. Moolenaar W.M. Schlessinger J. J. Biol. Chem. 2001; 276: 20130-20135Google Scholar). However, no information is available on the role of Pyk2 and the nature of its interaction with Src and EGF-R during receptor stimulation by GnRH. The present studies have identified a signaling cascade that mediates GnRH-induced ERK1/2 phosphorylation in immortalized GnRH neurons (GT1–7 cells) and is dependent on receptor-mediated activation of PKC, Src, Pyk2, and the EGF-R. GnRH was obtained from Peninsula Laboratories, Inc. (Belmont, CA), EGF was from Invitrogen, and pertussis toxin was from List Biological Laboratories. Protein assay kits were from Pierce. ERK1/2 and anti-phospho-ERK1/2 (Thr202/Tyr204) antibodies were from New England Biolabs, and secondary antibodies conjugated to horseradish peroxidase were from KPL. Antibodies against Src, EGF-R, phospho-EGF-R (Tyr1173), and phosphotyrosine (PY20) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-Pyk2 (Tyr402) was from either Calbiochem orBIOSOURCE International, and anti-phospho-EGF-R (Tyr1068) was from BIOSOURCEInternational. Mouse monoclonal hemagglutinin tag antibody was from Covance Babco (Berkeley, CA). AG1478, Go6983, Ro318220, PP2, BAPTA, PMA, and wortmannin were from Calbiochem, and antibodies against PKC isoforms and Pyk2 were from Transduction Laboratories. LipofectAMINE was from Invitrogen. The Pyk2, dominant negative Pyk2, constitutively active Src, and Csk constructs were provided by Dr. Zvi Naor (University of Tel Aviv). PKC isoform-specific dominant negative and constitutively active constructs tagged with the hemagglutinin epitope were prepared as previously described (32Soh J-W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Google Scholar). Western blotting reagents and ECL were obtained from Amersham Biosciences or Pierce. GT1–7 neurons donated by Dr. Richard Weiner (University of California, San Francisco) were grown in culture medium consisting of 500 ml of Dulbecco's modified Eagle's medium containing 0.584 g/liter l-glutamate and 4.5 g/liter glucose, mixed with 500 ml of F-12 medium containing 0.146 g/liter l-glutamate, 1.8 g/liter glucose, 100 μg/ml gentamicin, 2.5 g/liter sodium carbonate, and 10% heat-inactivated fetal calf serum. DNA transfections were performed with LipofectAMINE according to the manufacturer's instructions. Cells were labeled for 24 h in inositol-free Dulbecco's modified Eagle's medium containing 20 μCi/ml [3H]inositol as previously described (5Krsmanovic L.Z. Mores N. Navarro C.E. Saeed S.A. Arora K.K. Catt K.J. Endocrinology. 1998; 139: 4037-4043Google Scholar) and then washed twice with inositol-free M199 medium and stimulated at 37 °C in the presence of 10 mm LiCl. The reactions were stopped with perchloric acid, inositol phosphates were extracted, and radioactivity was measured by liquid scintillation γ-spectrometry. Serum-starved GT1–7 cells were treated with either PMA or GnRH for the times indicated and then washed twice with ice-cold PBS and collected in homogenization buffer containing 25 mm Tris·HCl, pH 7.4, 2 mm EDTA, 10 mm β-mercaptoethanol, 10% glycerol, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml each of aprotinin and leupeptin. After they were kept on ice for 10 min, cells were homogenized with 25 strokes of a Dounce homogenizer. Nuclei and unbroken cells were pelleted by centrifugation at 500 g for 5 min, and the supernatant was centrifuged at 100,000 × g for 30 min. The high speed supernatant constituted the cytosolic fraction. The pellet was washed three times and extracted in ice-cold homogenization buffer containing 1% Triton X-100 for 30 min. The Triton-soluble component (membrane fraction) was separated from the insoluble material (cytoskeletal fraction) by centrifugation at 100,000 × g for 20 min. After treatment with inhibitors and drugs, cells were placed on ice, washed twice with ice-cold PBS, lysed in lysis buffer (50 mm Tris, pH 8.0, 150 mmNaCl, 1 mm NaF, 0.25% sodium deoxycholate, 1 mm EDTA, 1% Nonidet P-40, 10 μg/ml aprotonin, 10 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml pepstain, and 1 mm 4-(2-aminoethyl)benzensulfonyl fluoride), and probe-sonicated (Sonifier cell disruptor). Solubilized lysates were clarified by centrifugation at 8000 × g for 10 min, precleared with agarose, and then incubated with specific antibodies and protein A- or G-agarose. The immunoprecipitates were collected, washed four times with lysis buffer, and dissolved in Laemmli buffer. After heating at 95 °C for 5 min, the samples were centrifuged briefly, and the supernatants were analyzed by SDS-PAGE on 8–16% gradient gels. Cells grown in six-well plates and at 60–70% confluence were serum-starved for 24 h before treatment at 37 °C with selected agents. The media were then aspirated, and the cells were washed twice with ice-cold PBS and lysed in 100 μl of Laemmli sample buffer. The samples were briefly sonicated, heated at 95 °C for 5 min, and centrifuged for 5 min. The supernatant was electrophoresed on SDS-PAGE (8–16%) gradient gels and transferred to polyvinylidene difluoride membranes. Blots were incubated overnight at 4 °C with primary antibodies and washed three times with TBST before probing with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Blots were then visualized with enhanced chemiluminescence reagent (AmershamBiosciences or Pierce) and quantitated with a laser-scanning densitometer. In some cases, blots were stripped and reprobed with other antibodies. GnRH treatment of GT1–7 cells caused transient stimulation of ERK1/2 that reached a peak at 5 min and declined thereafter toward the basal level over 30 min (Fig. 1 A). GnRH-induced ERK1/2 activation was concentration-dependent over the 0.2–200 nm range and was abolished by the GnRH receptor antagonist, [d-pGlu1,d-Phe,d-Trp(3,6)]GnRH (Fig. 1, B and C). GnRH receptors are primarily coupled to Gq/11 proteins, but some of the physiological actions of GnRH are known to occur through activation of Gsor Gi proteins (5Krsmanovic L.Z. Mores N. Navarro C.E. Saeed S.A. Arora K.K. Catt K.J. Endocrinology. 1998; 139: 4037-4043Google Scholar, 33Kaiser U.B. Conn P.M. Chin W.W. Endocr. Rev. 1997; 18: 46-70Google Scholar). In GT1–7 neurons, nanomolar GnRH concentrations cause marked elevation of inositol phosphate production through Gq-mediated activation of phospholipase C and also stimulate cAMP production. Higher concentrations of GnRH (0.1–1 μm) reduce intracellular cAMP levels in a pertussis toxin-sensitive manner, suggesting that GnRH activates a Giprotein in GT1–7 cells (34Krsmanovic L.Z. Mores N. Navarro C.E. Tomic M. Catt K.J. Mol. Endocrinol. 2001; 15: 429-440Google Scholar). Consistent with this, pertussis toxin had a modest inhibitory effect (∼30%) on GnRH-induced ERK activation, suggesting partial involvement of Gi protein(s) in MAPK signaling in GT1–7 cells (Fig. 1 D). The roles of PKC and Ca2+ in agonist-stimulated activation of ERK1/2 were evaluated in studies with PKC inhibitors and the Ca2+ chelators BAPTA-2AM and EGTA. GnRH-induced ERK1/2 activation was found to be highly PKC-dependent and was abolished by the PKC inhibitors Ro318220 and Go6983 (Fig. 2 A). These inhibitors had no effect on ERK1/2 activation induced by basic fibroblast growth factor (bFGF) (Fig. 2 B) or isoproterenol, a β2-adrenoreceptor agonist (data not shown). Consistent with its critical role in GnRH action, depletion of PKC by prolonged PMA treatment (1 μm for 16 h) abolished agonist-induced ERK1/2 activation (Fig. 2 C). However, ERK1/2 activation by GnRH was less sensitive to Ca2+ chelation by EGTA and BAPTA (Fig. 2 D). Consistent with this, the PKC activator, PMA, was much more effective than the Ca2+ionophore, ionomycin, in eliciting ERK1/2 activation (Fig. 2 E). These findings suggest that GnRH receptor-mediated ERK1/2 activation in GT1–7 cells is predominantly dependent on PKC. A major role of PKC in GnRH-induced ERK activation has also been reported in other cell types (8Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Google Scholar, 9Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Google Scholar, 10Grosse R. Roelle S. Herrlich A. Hohn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Google Scholar), but little information is available about the involvement of specific PKC isoforms in this cascade. GT1–7 cells were found to contain several immunoreactive PKC isoforms, including α, δ, ε, and λ. Overnight PMA stimulation (2 μm) caused down-regulation of PKCα, -δ, and -ε but not PKCλ (Fig. 3 A). Among the PMA-sensitive PKC isoforms, only PKCα and -ε were translocated from cytosol to the cell membrane during treatment with GnRH and PMA (Fig. 3 B). These effects of GnRH and PMA were specific, since no changes in the levels of ERK1/2 and Na+/K+-ATPase were found in cytosol and membranes, respectively (Fig. 3 C). The predominant role of PKCα in GnRH-induced ERK activation was confirmed in studies with constitutively active and dominant negative mutants of PKCα. These results showed that GnRH-induced ERK1/2 phosphorylation was attenuated by dominant negative PKCα (dnPKCα; Fig. 4) and was increased with transfection of constitutively active PKCα (data not shown).Figure 4The effects of dominant negative PKC α on ERK1/2 activation by GnRH.A, expression of native and transfected dominant negative PKCα (dnPKCα) in GT1–7 cells transfected with plasmid DNA (2 and 5 μg) encoding dominant negative PKCα. Cells were washed twice with ice-cold PBS and lysed in Laemmli sample buffer before loading onto 8–16% gradient gels for SDS-PAGE analysis. The expression of PKC constructs was detected using antibody against the hemagglutinin epitope with which these mutant proteins are tagged. Whereas conventional antibody against PKCα detects both native and exogenous dominant negative PKC proteins, the hemagglutinin antibody detects only the product of transfected dominant negative PKC with no immunoreactivity in the nontransfected (NT) cells.B, effects of overexpression of dominant negative PKCα on GnRH-induced ERK1/2 phosphorylation (ERK1/2-P). Serum-starved cells were stimulated with GnRH (200 nm for 5 min) and then washed twice with ice-cold PBS and lysed in Laemmli sample buffer before loading onto 8–16% gradient gels for SDS-PAGE analysis. The quantitated data are shown in the lower panel(n = 4).View Large Image Figure ViewerDownload (PPT) It is well established that transactivation of receptor tyrosine kinases such as the EGF-R contributes to GPCR-mediated ERK1/2 activation in certain cell types (14Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Google Scholar, 22Gschwind A. Zwick E. Prenzel N. Leserer M. Ullrich A. Oncogene. 2001; 20: 1594-1600Google Scholar). However, studies on the role of the EGF-R in GnRH action have not given consistent results (9Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Google Scholar, 10Grosse R. Roelle S. Herrlich A. Hohn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Google Scholar). Thus, whereas Grosse et al. (10Grosse R. Roelle S. Herrlich A. Hohn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Google Scholar) implicated transactivation of the EGF-R in GnRH-induced stimulation of ERK1/2 phosphorylation in αT3–1 pituitary gonadotrophs, Benard et al. (9Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Google Scholar) subsequently reported that the major pathway of ERK1/2 activation was through PKC and activation of Raf-1 and did not involve the EGF-R. Since GT1–7 cells express receptors for both EGF and GnRH, we evaluated the role of the EGF-R in GnRH-induced MAPK signaling. In this cell type, EGF, like GnRH, caused transient activation of ERK1/2 (Fig. 5 A). As expected, the selective EGF-R kinase inhibitor, AG1478, blocked the ERK1/2 activation induced by EGF (Fig. 5 B). EGF stimulation caused rapid and marked phosphorylation of the EGF-R at Tyr1173 in a time- and concentration-dependent manner (Fig. 5, C andD). Our data suggest a potential role of PKC in GnRH-induced ERK1/2 activation. To determine whether PKC acts upstream or downstream of the EGF-R, we examined the effect of PKC inhibition on EGF-induced ERK1/2 activation. Whereas PKC depletion by prolonged PMA treatment or PKC inhibitors abolished the effects of PMA and GnRH, it had no effect on EGF responses (Fig. 5, E and F). These data indicate that EGF-induced ERK1/2 activation is PKC-independent and that PKC acts upstream of the EGF-R during GnRH signaling in GT1–7 cells. To examine the involvement of EGF-R in GnRH-induced ERK1/2 activation in GT1–7 cells, cells were pretreated with AG1478 and stimulated with GnRH (200 nm for 5 min). As shown in Fig. 6 A, GnRH-stimulated ERK1/2 phosphorylation was also abolished by AG1478, indicating its absolute dependence on transactivation of the EGF-R. The inhibitory action of AG1478 on GnRH-induced ERK1/2 activation was selective and did not affect bFGF-stimulated ERK1/2 phosphorylation (Fig. 6 B). Consistent with the role of EGF-R in GnRH signaling, GnRH also stimulated phosphorylation of the EGF-R as measured with anti-phosphopeptide antibodies that recognize the phosphorylated molecule at Tyr1173 or Tyr1168 (Fig. 6 C), the major sites of Src kinase phosphorylation (35Wright J.D. Reuter C.W. Weber M.J. Biochim. Biophys. Acta. 1996; 1312: 85-93Google Scholar) and Grb2 binding (36Zwick E. Wallasch C. Daub H. Ullrich A. J. Biol. Chem. 1999; 274: 20989-20996Google Scholar), respectively. These data demonstrate that transactivation and phosphorylation of the EGF-R are essential for GnRH signaling through ERK1/2 in GT1–7 cells. Since GnRH-induced ERK1/2 activation is primarily dependent on PKC and GnRH causes PKC activation through generation of diacylglycerol (8Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Google Scholar, 9Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Google Scholar, 10Grosse R. Roelle S. Herrlich A. Hohn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Google Scholar), we investigated the effects of PMA on this cascade. The results revealed that PMA caused marked ERK1/2 activation that was abolished by prior PKC depletion (as shown above in Fig. 5 D) and by PKC inhibitors, Ro318220 and Go6983 (Fig. 7, A and B). To determine whether PMA mimics the effects of GnRH with respect to EGF-R transactivation, GT1–7 cells were treated with AG1478 and stimulated with PMA. As shown in Fig. 7 C, PMA-induced ERK1/2 activation was extinguished in a dose-dependent manner by the EGF-R kinase inhibitor, AG1478, indicating that GnRH-induced ERK1/2 activation occurs through EGF-R transactivation in a PKC-dependent manner. Since there is no consensus on the types of intermediate proteins involved during GPCR-induced transactivation of the EGF-R (14Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Google Scholar,36Zwick E. Wallasch C. Daub H. Ullrich A. J. Biol. Chem. 1999; 274: 20989-20996Google Scholar, 37Shah B.H. Catt K.J. Mol. Pharmacol. 2002; 61: 343-351Google Scholar), we examined the roles of Src and Pyk2 in GnRH-induced EGF-R phosphorylation and ERK activation. In GT1–7 cells, the highly selective Src kinase inhibitor, PP2, and the Src negative regulatory kinase, Csk, attenuated the activation of ERK1/2 by GnRH (Fig. 8, A and B). Similarly, Src inhibition abolished the effect of PMA on ERK1/2 activation (Fig. 8 C). In contrast, Src inhibition and Csk had no effect on EGF-induced ERK1/2 responses (Fig. 8 D). These data suggest that Src has a critical role in GnRH-induced activation of the EGF-R and ERK1/2. Since our data show that both PKC and Src act upstream of EGF-R, we examined the interaction between PKC and Src. As shown in Fig. 8 E, GnRH stimulation increased the association of PKCα and -ε with Src. A role for the nonreceptor proline-rich tyrosine kinase, Pyk2, in ERK1/2 activation by some GPCRs has been shown (26Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owasa K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Google Scholar, 27Matsubara H. Shibasaki Y. Okigaki M. Mori Y. Masaki H. Kosaki A. Tsutsumi Y. Biochem. Biophys. Res. Commun. 2001; 282: 1085-10891Google Scholar, 28Sorokin A. Kozlowski P. Graves L. Philip A. J. Biol. Chem. 2001; 276: 21521-21528Google Scholar, 29Iwasaki H. Shichiri M. Marumo F. Hirata Y. Endocrinology. 2001; 142: 564-572Google Scholar, 30Keely S.J. Calandrella S.O. Barrett K.E. J. Biol. Chem. 2000; 275: 12619-12625Google Scholar). However, nothing is known about the role of Pyk2 in GnRH signaling. GnRH stimulation of GT1–7" @default.
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