The GnRH decapeptide is the primary regulatory factor in the neuroendocrine control of reproduction and is released in an episodic manner from hypothalamic GnRH neurons, which we previously found to express GnRH receptors. The hypothalamic control of pituitary and gonadal function is dependent on the pulsatile secretion of GnRH from the median eminence into the descending portal vessels to the anterior pituitary gland. We observed that GnRH agonists exert both stimulatory and inhibitory actions on GnRH secretion in GnRH neurons and GT1-7 cells, due to GnRH receptor-mediated activation of multiple G proteins in a time- and dose-dependent manner. The resulting differential regulation of PLC/InsP3/Ca2+ and adenylyl cyclase/cAMP signaling pathways, and also of GnRH neuronal firing, regulates the frequency and amplitude of pulsatile GnRH release. Such actions can result from agonist-regulated sequential coupling of the activated GnRH receptor to Gq, Gs, and Gi proteins, or from the activation of multiple receptor subtypes (eg, 5-HT1A, 5-HT2C, 5-HT4, and 5-HT7) by serotonin, the non-selective 5-HT receptor agonist. The ensuing signaling responses, in conjunction with their modulation of the spontaneous electrical activity of the GnRH neuron, determine the pulsatile mode of neuropeptide secretion that is characteristic of GnRH neuronal function in vivo and in vitro. ? ? A similar paradigm occurs during activation of the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor (LHR) in cultured hypothalamic cells and GT1-7 cells. In the latter cells, treatment with LH or hCG transiently stimulates and subsequently inhibits cAMP production and pulsatile GnRH release. The delayed impairment of cAMP signaling and episodic GnRH release in GT1-7 cells is prevented by pertussis toxin (PTX). These, and the LH/hCG-induced release of membrane-bound Gs and Gi3 subunits, demonstrate differential G protein coupling to the LHR. AP firing in identified GnRH neurons initially increased and then progressively decreased during LH treatment. The inhibitory action of LH on AP firing was also prevented by PTX. RT-PCR analysis of GT1-7 neurons revealed the expression of G protein-gated inwardly rectifying potassium (GIRK) channels in these cells. LH-induced currents were inhibited by PTX and were identified by electrophysiological studies as GIRK currents. These responses indicate that agonist-stimulation of endogenous LHR expressed in GnRH neurons activates GIRK channels, leading to suppression of membrane excitability and inhibition of AP firing. These findings demonstrate the importance of GIRK channel regulation in gonadotropin-induced inhibition of pulsatile GnRH release. This Gi-mediated mechanism could also account for the suppression of pituitary function in pregnancy and during ectopic hCG production.? ? Activation of GnRH receptors stimulation also promotes proliferative signals and cell survival in hypothalamic GT1-7 cells. Both GnRH and EGF caused rapid phosphorylation of CREB and also BAD, a member of the Bcl2 family. The EGF-R antagonist, AG1478, attenuated such phosphorylation by GnRH and EGF. The stimulatory effects of GnRH, but not that of EGF, were abolished by inhibition of PKC and Src, consistent with their critical role upstream of the EGF-R. These effects of GnRH were also elicited by activation of PKC. Consistent with the prosurvival and mitogenic effects of PI 3-kinase/Akt (PI3K/Akt) downstream of the EGF-R, inhibition of PI3K diminished the activation of these proteins following stimulation with GnRH, EGF, and PMA. Over-expression of dominant negative Akt attenuated agonist-induced phosphorylation of BAD, but not of ERK1/2 and CREB. Also, over-expression of wild-type RSK-1 caused enhanced basal as well as agonist-induced phosphorylation of CREB and BAD, indicating a critical role of RSK-1 in activating cytosolic as well as nuclear proteins. These data have revealed novel signaling mechanisms of GnRH-induced phosphorylation of CREB and BAD in GT1-7 neurons through transactivation of the EGF-R. ? ? Adrenergic receptors (ARs) are involved in the regulation of GnRH release from native and immortalized hypothalamic (GT1-7) neurons, but their signaling mechanisms and their functional significance have not been defined. In GT1-7 cells, the alpha1-AR agonist, phenylephrine (Phe), caused PKC-dependent transactivation of the EGF-R and phosphorylation of MAP kinases (ERK1&2), due to shedding of HB-EGF by matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP with CRM197, or of HB-EGF by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These findings indicate that activation of the alpha1-AR causes phosphorylation of ERK1/2 through PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.? ? Agonist-activation of many GPCRs causes phosphorylation of MAP kinases through transactivation of the EGF-R, leading to increased cell survival and growth, motility, and migration. Phosphatidylinositol 3-kinase (PI3K) is one of the major cell survival signaling molecules activated by EGF-R stimulation. The extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation was determined by analysis of the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. GT1-7 neuronal cells express endogenous GnRH-R, activation of which causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1, lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-Rs caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF-R and PI3K, respectively. These data show that the dependence of GPCR-induced ERK1/2 phosphorylation on PI3K is variable in specific cell types, and that the involvement of PI3K during ERK1/2 activation does not necessarily result from agonist-induced transactivation of the EGF-R.
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