Abstract
Gonadotropin-releasing hormone receptors (GnRHR) mediate activation and nuclear translocation of the extracellular signal regulated kinases 1 and 2 (ERK) by phosphorylation on the TEY motif. This is necessary for GnRH to initiate transcriptional programmes controlling fertility, but mechanisms that govern ERK targeting are unclear. Using automated microscopy to explore ERK regulation in single cells, we find that GnRHR activation induces marked redistribution of ERK to the nucleus and that this effect can be uncoupled from the level of TEY phosphorylation of ERK. Thus, 5 min stimulation with 100 nM GnRH increased phospho-ERK levels (from 89±34 to 555±45 arbitrary fluorescence units) and increased the nuclear:cytoplasmic (N:C) ERK ratio (from 1.36±0.06 to 2.16±0.05) in the whole cell population, but it also significantly increased N:C ERK in cells binned according to phospho-ERK levels. This phosphorylation unattributable component of the ERK translocation response occurs at a broad range of GnRHR expression levels, in the presence of tyrosine phosphatase and protein synthesis inhibitors, and in ERK mutants unable to undergo catalytic activation. It also occurred in mutants incapable of binding the DEF (docking site for ERK, F/Y-X-F/Y-P) domains found in many ERK binding partners. It was however, reduced by MEK or PKC inhibition and by mutations preventing TEY phosphorylation or that abrogate ERK binding to D (docking) domain partners. We therefore show that TEY phosphorylation of ERK is necessary, but not sufficient for the full nuclear localization response. We further show that this “phosphorylation unattributable” component of GnRH-mediated ERK nuclear translocation requires both PKC activity and association with partner proteins via the D-domain.
Highlights
The gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide that is the master control hormone in reproduction [1]
Most established culture models of gonadotrope cells are derived by SV40 T antigen-mediated immortalization [38]. As this is known to affect phosphatases that control the duration of ERK responses [39], we opted to study ERK signalling in a model HeLa cell system, using Ad to express murine Gonadotropin-releasing hormone receptors (GnRHR) within physiological ranges of expression
We first determined the time course of ERK activation by immunoblotting for TEY phosphorylated ERK1 and/or ERK2 and total ERK in HeLa cells transduced with mGnRHR and stimulated for 0 or 5– 240 min with 100 nM GnRH or 1 mM PDBu (Fig. 1A)
Summary
The gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2) that is the master control hormone in reproduction [1]. GnRH is secreted in a pulsatile fashion by the hypothalamus and acts on Gq/11-coupled seven transmembrane (7TM) GnRH receptors (GnRHRs) in gonadotrope cells of the pituitary. This causes the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRHR activation initiates several intracellular signalling cascades in gonadotropes, but activation of the ERK extracellular signal-regulated kinase) MAPK (mitogen-activated protein kinase) cascade is responsible for a large proportion of the biological effects elicited by GnRH [1,2,3]. GnRH can cause ERK cascade activation through a variety of signalling routes, such as activation of protein kinase C (PKC) isozymes, and/or transactivation of the epidermal growth factor receptor (EGFR). Activated Raf can phosphorylate and activate the cytosolic kinases, MEK
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