Pulsatile and Sustained Gonadotropin-releasing Hormone (GnRH) Receptor Signaling

Abstract

Gonadotropin-releasing hormone (GnRH) acts via 7 transmembrane region receptors on gonadotrophs to stimulate synthesis and secretion of the luteinizing hormone and follicle-stimulating hormone. It is secreted in pulses, and its effects depend on pulse frequency, but decoding mechanisms are unknown. Here we have used (nuclear factor of activated T-cells 2 (NFAT2)-emerald fluorescent protein) to monitor GnRH signaling. Increasing [Ca2+]i causes calmodulin/calcineurin-dependent nuclear NFAT translocation, a response involving proteins (calmodulins and NFATs) that decode frequency in other systems. Using live cell imaging, pulsatile GnRH caused dose- and frequency-dependent increases in nuclear NFAT2-emerald fluorescent protein, and at low frequency, translocation simply tracked GnRH exposure (albeit with slower kinetics). At high frequency (30-min intervals), failure to return to basal conditions before repeat stimulation caused integrative tracking, illustrating how the relative dynamics of up- and downstream signals can increase efficiency of GnRH action. Mathematical modeling predicted desensitization of GnRH effects on [Ca2+]i and that desensitization would increase with dose, frequency, and receptor number, but no such desensitization was seen in HeLa and/or LβT2 cells possibly because pulsatile GnRH did not reduce receptor expression (measured by immunofluorescence). GnRH also caused dose- and frequency-dependent activation of αGSU, luteinizing hormone β, and follicle-stimulating hormone β luciferase reporters, effects that were blocked by calcineurin inhibition. Pulsatile GnRH also activated an NFAT-responsive luciferase reporter, but this response was directly related to cumulative pulse duration. This together with the lack of desensitization of translocation responses suggests that NFAT may mediate GnRH action but is not a genuine decoder of GnRH pulse frequency.

Signaling Responses to Pulsatile Gonadotropin-releasing Hormone in LβT2 Gonadotrope Cells

“…Although NFAT activation is thought to be dependent on the calcium activation of calcineurin, and indeed complete removal of calcium did inhibit the translocation, Armstrong et al (27) did not observe desensitization of NFAT translocation. NFAT can also be activated by cAMP signaling in cardiomyocytes and osteoclasts, however, so the observed NFAT translocation may reflect cAMP rather than calcium signaling (28,29).…”

“…A recent publication reported the rapid translocation of NFAT-EFP to the nucleus in response to multiple pulses of GnRH in real time in transfected HeLa and L␤T2 cells (27). Although NFAT activation is thought to be dependent on the calcium activation of calcineurin, and indeed complete removal of calcium did inhibit the translocation, Armstrong et al (27) did not observe desensitization of NFAT translocation.…”

Stimulus-induced uncoupling of extracellular signal-regulated kinase phosphorylation from nuclear localization is dependent on docking domain interactions

“…K52R, T183-Y185A, Y261A and D319N mutations were introduced using a QuikChange PCR-based mutagenesis kit (Stratagene, Amsterdam, The Netherlands) and the following primers: 5Ј-CAA AGT TCG AGT TGC TAT CAG GAA AAT CAG TCC TTT TGA GC-3Ј, 5Ј-TGA TCA TAC AGG GTT CTT GGC AGA GGC TGT AGC CAC GCG TTG GTA C-3Ј, 5Ј-AAT TTA AAA GCT AGA AAC GCT TTG CTT TCT CTC CCG CAC-3Ј and 5Ј-GCA GTA TTA TGA CCC AAG TAA TGA GCC CAT TGC TGA AGC-3Ј, along with antisense primers according to the manufacturer's recommendations and using the pacAd5CMV ERK2-GFP vector as the template. Adenovirus (Ad) Egr-1-luciferase and Ad CMV -galactosidase reporter shuttle vectors were constructed as described previously (Caunt et al, 2008a;Armstrong et al, 2010). Viruses were generated from shuttle vectors as described previously (Anderson et al, 2000;Caunt et al, 2006b).…”

Calcineurin Mediates the Gonadotropin-Releasing Hormone Effect on Expression of Both Subunits of the Follicle-Stimulating Hormone through Distinct Mechanisms

“…These transcription factors are phosphorylated in the cytoplasm and are activated by calcineurin-mediated dephosphorylation, which allows their translocation into the nucleus. Interestingly, one of the NFAT isoforms, NFAT2, was used as a reporter for GnRH-activated calcium signaling in L␤T2 cells, and the overexpressed fluorescent protein was translocated to the nucleus in a dose-and frequency-dependent manner (2,3).…”