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Perrett R.M.,University of Bristol | Voliotis M.,University of Bristol | Voliotis M.,Medical Research Council Biomedical Informatics Fellowship | Armstrong S.P.,University of Bristol | And 4 more authors.
Journal of Biological Chemistry | Year: 2014

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses that stimulate synthesis and secretion of pituitary gonadotropin hormones and thereby mediate control of reproduction. It acts via G-protein-coupled receptors to stimulate effectors, including ERK. Information could be encoded in GnRH pulse frequency, width, amplitude, or other features of pulse shape, but the relative importance of these features is unknown. Here we examine this using automated fluorescence microscopy and mathematical modeling, focusing on ERK signaling. The simplest scenario is one in which the system is linear, and response dynamics are relatively fast (compared with the signal dynamics). In this case integrated system output (ERK activation or ERK-driven transcription) will be roughly proportional to integrated input, but we find that this is not the case. Notably, we find that relatively slow response kinetics lead to ERK activity beyond the GnRH pulse, and this reduces sensitivity to pulse width. More generally, we show that the slowing of response kinetics through the signaling cascade creates a system that is robust to pulse width. We, therefore, show how various levels of response kinetics synergize to dictate system sensitivity to different features of pulsatile hormone input. We reveal the mathematical and biochemical basis of a dynamic GnRH signaling system that is robust to changes in pulse amplitude and width but is sensitive to changes in receptor occupancy and frequency, precisely the features that are tightly regulated and exploited to exert physiological control in vivo. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Armstrong S.P.,University of Bristol | Caunt C.J.,University of Bath | Fowkes R.C.,Endocrine Signaling Group | Tsaneva-Atanasova K.,University of Bristol | McArdle C.A.,University of Bristol
Journal of Biological Chemistry | Year: 2010

Gonadotropin-releasing hormone (GnRH) acts via G-protein-coupled receptors on gonadotrophs to stimulate synthesis and secretion of 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 an extracellular signal regulated kinase-green fluorescent protein (ERK2-GFP) reporter to monitor GnRH signaling. GnRH caused dose-dependent ERK2-GFP translocation to the nucleus, providing a live-cell readout for activation. Pulsatile GnRH caused dose- and frequency-dependent ERK2-GFP translocation. These responses were rapid and transient, showed only digital tracking, and did not desensitize under any condition tested (dose, frequency, and receptor number varied). We also tested for the effects of cycloheximide (to prevent induction of nuclear-inducible MAPK phosphatases) and used GFP fusions containing ERK mutations (D319N, which prevents docking domain-dependent binding to MAPK phosphatases, and K52R, which prevents catalytic activity). Thesemanipulationshadlittleornoeffectonthetranslocation responses, arguing against a role for MAPK phosphatases or ERKmediated feedback in shaping ERK activation during pulsatile stimulation. GnRH also caused dose- and frequency-dependent activation of the α-gonadotropin subunit-, luteinizing hormone β-, and follicle-stimulating hormone β- luciferase reporters, and the latter response was inhibited by ERK1/2 knockdown. Moreover, GnRH caused frequency-dependent activation of an Egr1-luciferase reporter, but the response was proportional to cumulative pulse duration. Our data suggest that frequency decoding is not due to negative feedback shaping ERK signaling in this model. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Chahal H.S.,Queen Mary, University of London | Trivellin G.,Queen Mary, University of London | Leontiou C.A.,Queen Mary, University of London | Alband N.,Queen Mary, University of London | And 14 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2012

Context: Somatotroph adenomas harboring aryl hydrocarbon receptor interacting protein (AIP) mutations respond less well to somatostatin analogs, suggesting that the effects of somatostatin analogs may be mediated by AIP. Objective: The objective of the investigation was to study the involvement of AIP in the mechanism of effect of somatostatin analogs. Design: In the human study, a 16-wk somatostatin analog pretreatment compared with no pretreatment. In the in vitro cell line study, the effect of somatostatin analog treatment or small interfering RNA (siRNA)/plasmid transfection were studied. Setting: The study was conducted at a university hospital. Patients: Thirty-nine sporadic and 10 familial acromegaly patients participated in the study. Intervention: Interventions included preoperative lanreotide treatment and pituitary surgery. Outcome: For the human study, GH and IGF-I levels, AIP, and somatostatin receptor staining were measured. For the cell line, AIP and ZAC1 (zinc finger regulator of apoptosis and cell cycle arrest) expression, metabolic activity, and clone formation were measured. Results: Lanreotide pretreatment reduced GH and IGF-I levels and tumor volume (all P < 0.0001). AIP immunostaining was stronger in the lanreotide-pretreated group vs. the surgery-only group (P < 0.001). After lanreotide pretreatment, the AIP score correlated to IGF-I changes in females (R = 0.68, P < 0.05). Somatostatin receptor staining was not reduced in samples with AIP mutations. In GH3 cells, 1 nM octreotide increased AIP mRNA and protein (both P < 0.01) and ZAC1 mRNA expression (P < 0.05). Overexpression of wild-type (but not mutant) AIP increased ZAC1 mRNA expression, whereas AIP siRNA knockdown reduced ZAC1 mRNA (both P < 0.05). The siRNA-mediated knockdown of AIP led to an increased metabolic activity and clonogenic ability of GH3 cells compared with cells transfected with a nontargeting control (both P < 0.001). Conclusion: These results suggest that AIP may play a role in the mechanism of action of somatostatin analogs via ZAC1 in sporadic somatotroph tumors and may explain their lack of effectiveness in patients with AIP mutations. Copyright © 2012 by The Endocrine Society. Source


Perrett R.M.,University of Bristol | Fowkes R.C.,Endocrine Signaling Group | Caunt C.J.,University of Bath | Tsaneva-Atanasova K.,University of Bristol | And 2 more authors.
Journal of Biological Chemistry | Year: 2013

Background: The mechanisms underlying acute ERK signaling are poorly understood. Results: Feedback influences basal and acutely stimulated ERK responses but does not render signaling kinetics robust to ERK concentration. Conclusion: Acute ERK response kinetics depend on ERK concentration and activation mechanism as well as feedback. Significance: ERK responses to transient stimulation can be gated by ERK concentration, and short-term activation appears distributive rather than processive. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Caunt C.J.,University of Bath | Perett R.M.,University of Bristol | Fowkes R.C.,Endocrine Signaling Group | McArdle C.A.,University of Bristol
PLoS ONE | Year: 2012

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. © 2012 Caunt et al. Source

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