The episodic secretory activity of the hypothalamic GnRH-producing neuronal network is essential for normal mammalian reproduction, and is both calcium-dependent and stimulated by cAMP. It is also regulated by agonist activation of the endogenous Gq-coupled GnRH receptor, which stimulates inositol phosphate/Ca2+ and cAMP signaling. Conversely, GnRH receptor antagonists inhibit activation of membrane-associated Gq and abolish pulsatile GnRH secretion. High GnRH concentrations cause receptor coupling to Gi and inhibit cAMP production, with reduction of membrane-associated alpha(s) and alpha(i3) levels. Conversely, membrane-associated alpha(i3) is increased by GnRH antagonist and pertussis toxin treatment, with concomitant loss of pulsatile GnRH secretion. Treatment with cholera toxin and 8-Br-cAMP amplifies episodic GnRH pulses but does not alter their frequency. These findings indicate that an agonist concentration-dependent switch in coupling of the GnRHR between specific G proteins modulates neuronal signaling via Gs/Gq stimulatory and Gi inhibitory mechanisms. The latter may also suppress GnRH neuronal firing and episodic secretion by regulating membrane ion currents. This autocrine mechanism serves as a timer to determine the frequency of pulsatile GnRH release by regulating Ca2+- and cAMP-dependent signaling and GnRH neuronal firing. An investigation of the ontogeny and function of this autoregulatory process in GnRH neurons derived from 13-day fetal rats revealed that immunocytochemically identified, laser-captured fetal rat hypothalamic GnRH neurons, and fetal GnRH neurons identified by differential interference contrast microscopy, coexpress mRNAs encoding GnRH and its type I receptor. Such fetal neurons exhibit spontaneous electrical activity that is increased by GnRH agonist treatment. This evoked response, as well as basal neuronal firing, are abolished by GnRH antagonists. GnRH stimulation elicits biphasic [Ca2+]i responses, and both basal and GnRH-stimulated [Ca2+]i levels are reduced by antagonist treatment. Perifused neuronal cultures release GnRH in a pulsatile manner that is highly dependent on extracellular Ca2+. GnRH pulse amplitude is increased by agonist stimulation and diminished by GnRH antagonists. These findings demonstrate that GnRH receptor-dependent activation of Ca2+ signaling and autocrine regulation of GnRH release are characteristics of early fetal GnRH neurons, and provides a mechanism for their endogenous gene expression and episodic GnRH secretion. These activities could also be essential for promoting the migration of embryonic GnRH neurons to the hypothalamus. Hypothalamic GnRH neurons and immortalized GT1-7 cells express cell membrane-associated and nuclear estrogen receptor (ER) alpha and beta isoforms. The cell-surface ERalpha receptor expressed in GnRH neurons undergoes high-affinity interactions with adenylyl cyclase inhibitory G proteins, modulates intracellular cAMP signaling, and regulates the GnRH secretory profile. The sensitivity of these interactions to picomolar estradiol concentrations suggests that this process represents a physiological negative feedback action of estrogen on the GnRH neuron. Picomolar estradiol concentrations cause rapid, sustained, and dose-dependent inhibition of cAMP production, whereas nanomolar estradiol concentrations increase cAMP production. Both of these opposing actions of estradiol are abolished by the ER antagonist, ICI 182,780. Estradiol-induced inhibition of cAMP production is also prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrate an estradiol-regulated interaction between ERalpha and Galphai3 that is prevented by ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increases the GnRH peak interval, shortens peak duration, and increases peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physiological estradiol levels causes activation of a Gi protein and modulates cAMP signaling and neuropeptide secretion. The signaling pathways of GPCRs include transactivation of receptor tyrosine kinases (RTKs) such as the EGFR, platelet-derived growth factor, neurotrophins and fibroblast growth factor. The complex cross-communication between GPCRs and RTKs utilizes arrays of signaling molecules that depend on cell context and the types of receptors activated. The differential involvement of RTKs and downstream signaling pathways activated in response to GPCR stimulation affect cell development, proliferation, differentiation, survival, and repair, as well as synaptic transmission. GnRH utilizes multiple signaling pathways to activate extracellularly regulated MAP kinases, such as ERK1/2, in normal and immortalized pituitary gonadotrophs and GT1-7 neurons, which express receptors for GnRH and epidermal EGF. In GT1-7 cells, GnRH and PMA cause rapid phosphorylation at Tyr 402 of the proline-rich tyrosine kinase, Pyk2, that is dependent on Ca2+ and activation of PKC. GnRH stimulation translocates PKCalpha and epsilon to the cell membrane and enhances the association of Src with activated PKC isoforms, Pyk2, and the EGF receptor. Inhibition of Src kinase, and dominant negative Pyk2, attenuates ERK1/2 activation by GnRH, but not by EGF, indicating 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 duration and magnitude of MAP kinase activation regulate gene expression and other specific intracellular responses in individual cell types. GnRH-induced activation of ERK1/2 by GnRH is transient in immortalized GT1-7 neurons, and neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus. However, both EGF and GnRH stimulated phosphorylation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1) at Ser360/Thr364, and caused its translocation into the nucleus. Thus, the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells, and transactivation of the ERFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain GPCRs. An analysis of the processes involved in such EGFR transactivation showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, HB-EGF, as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGFR and subsequently of Shc, ERK1/2, and its dependent protein, RSK-1, was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with a neutralizing antibody, or the selective inhibitor CRM197, attenuated signals generated by GnRH and PMA but not those stimulated by EGF. In contrast, phosphorylation of the EGFR, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resemble those of GnRH and EGF in terms of the phosphorylation of the EGFR, Shc, ERK1/2, and RSK-1, as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor overexpression of Csk and dominant negative Pyk2, impaired HB-EGF-induced responses. In contrast, HEK293 cells expressing GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These findings demonstrate that GnRH-induced transactivation of the EGF-R, and the subsequent ERK1/2 phosphorylation, result from ectodomain shedding of HB-EGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.
NIH ExPorter
NIH ExPorter