Abstract
Numerous external stimuli, including G protein-coupled receptor agonists, cytokines, growth factors, and steroids activate mitogen-activated protein kinases (MAPKs) through phosphorylation of the epidermal growth factor receptor (EGF-R). In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. An analysis of the mechanisms involved in this process showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, heparin binding epidermal growth factor (HB-EGF), as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, p90RSK-1 (p90 ribosomal S6 kinase 1 or RSK-1), was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with the selective inhibitor CRM197 or a neutralizing antibody attenuated signals generated by GnRH and phorbol 12-myristate 13-acetate, but not those stimulated by EGF. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resembled those of GnRH and EGF in terms of the phosphorylation of EGF-R, Shc, ERK1/2, and RSK-1 as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor the overexpression of negative regulatory Src kinase and dominant negative Pyk2 had any effect on HB-EGF-induced responses. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.
Highlights
A major potential mechanism of the cross-communication between G protein-coupled receptor (GPCR) and receptor tyrosine kinases (RTKs) is the activation of metalloproteases that cause proteolytic cleavage of the transmembrane, proheparin-binding EGF precursor to yield the soluble ligand HBEGF, which binds to and activates the epidermal growth factor receptor (EGF-R) [6]
In GT1-7 neuronal cells, which express endogenous receptors for both Gonadotropin-releasing hormone (GnRH) and EGF, activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by either of these ligands was abrogated by the selective EGF-R kinase inhibitor AG1478, consistent with the dependence of GnRHinduced ERK1/2 activation upon transactivation of the EGF-R (Fig. 1A)
These findings demonstrate the specificity of GM6001 and indicate the dependence of GnRH signaling on metalloprotease action upstream of the EGF-R
Summary
Vol 279, No 1, Issue of January 2, pp. 414 –420, 2004 Printed in U.S.A. Neuropeptide-induced Transactivation of a Neuronal Epidermal Growth Factor Receptor Is Mediated by Metalloprotease-dependent Formation of Heparin-binding Epidermal Growth Factor*. In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells. Recent studies have suggested that MAPK activation by external stimuli, such as G protein-coupled receptor (GPCR) agonists, cytokines, growth hormones, steroids and environmental stresses, occurs through transactivation of receptor tyrosine kinases (RTKs), in particular the epidermal growth factor receptor (EGF-R). HBEGF mimics the effects of EGF as shown by tyrosine phosphorylation of the EGF-R and the activation of downstream signaling molecules in GT1-7 cells
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