Stephen P. Armstrong

Spatiotemporal regulation of ERK2 by dual specificity phosphatases.

Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.

Encoding and Decoding Mechanisms of Pulsatile Hormone Secretion

Ultradian pulsatile hormone secretion underlies the activity of most neuroendocrine systems, including the hypothalamic‐pituitary adrenal (HPA) and gonadal (HPG) axes, and this pulsatile mode of signalling permits the encoding of information through both amplitude and frequency modulation. In the HPA axis, glucocorticoid pulse amplitude increases in anticipation of waking, and, in the HPG axis, changing gonadotrophin‐releasing hormone pulse frequency is the primary means by which the body alters its reproductive status during development (i.e. puberty). The prevalence of hormone pulsatility raises two crucial questions: how are ultradian pulses encoded (or generated) by these systems, and how are these pulses decoded (or interpreted) at their target sites? We have looked at mechanisms within the HPA axis responsible for encoding the pulsatile mode of glucocorticoid signalling that we observe in vivo. We review evidence regarding the ‘hypothalamic pulse generator’ hypothesis, and describe an alternative model for pulse generation, which involves steroid feedback‐dependent endogenous rhythmic activity throughout the HPA axis. We consider the decoding of hormone pulsatility by taking the HPG axis as a model system and focussing on molecular mechanisms of frequency decoding by pituitary gonadotrophs.

Pulsatile and sustained gonadotropin-releasing hormone (GnRH) receptor signaling: Does the Ca2+/NFAT signaling pathway decode GnRH pulse frequency?

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.

Agonist-induced internalization and downregulation of gonadotropin-releasing hormone receptors.

Gonadotropin-releasing hormone (GnRH) acts via seven transmembrane receptors to stimulate gonadotropin secretion. Sustained stimulation desensitizes GnRH receptor (GnRHR)-mediated gonadotropin secretion, and this underlies agonist use in hormone-dependent cancers. Since type I mammalian GnRHR do not desensitize, agonist-induced internalization and downregulation may underlie desensitization of GnRH-stimulated gonadotropin secretion; however, research focus has recently shifted to anterograde trafficking, with the finding that human (h)GnRHR are mostly intracellular. Moreover, there is little direct evidence for agonist-induced trafficking of hGnRHR, and whether or not type I mammalian GnRHR show agonist-induced internalization is controversial. Here we use automated imaging to monitor expression and internalization of hemagglutinin (HA)-tagged hGnRHRs, mouse (m) GnRHR, Xenopus (X) GnRHRs, and chimeric receptors (hGnRHR with added XGnRHR COOH tails, h.XGnRHR) expressed by adenoviral transduction in HeLa cells. We find that agonists stimulate downregulation and/or internalization of mGnRHR and XGnRHR, that GnRH stimulates trafficking of hGnRHR and can stimulate internalization or downregulation of hGnRHR when steps are taken to increase cell surface expression (addition of the XGnRHR COOH tail or pretreatment with pharmacological chaperone). Agonist effects on internalization (of h.XGnRHR) and downregulation (of hGnRHR and h.XGnRHR) were not mimicked by a peptide antagonist and were prevented by a mutation that prevents GnRHR signaling, demonstrating dependence on receptor signaling as well as agonist occupancy. Thus agonist-induced internalization and downregulation of type I mammalian GnRHR occurs in HeLa cells, and we suggest that the high throughput imaging systems described here will facilitate study of the molecular mechanisms involved.

Gonadotropin-Releasing Hormone and Protein Kinase C Signaling to ERK: Spatiotemporal Regulation of ERK by Docking Domains and Dual-Specificity Phosphatases

Activated ERK translocates to the nucleus to regulate transcription. Spatiotemporal aspects of this response dictate biological consequences and are influenced by dual-specificity phosphatases (DUSPs) that can scaffold and dephosphorylate ERK. In HeLa cells, GnRH causes transient and protein kinase C (PKC)-dependent ERK activation, but termination mechanisms are unknown. We now explore DUSP roles using short inhibitory RNA to knock down endogenous ERK, adenoviruses to express GnRH receptors and add-back ERK2-GFP, and automated microscopy to monitor ERK location and activation. GnRH caused rapid and transient increases in dual phosphorylated ERK2 (ppERK2) and nuclear to cytoplasmic ERK2-green fluorescent protein (GFP) ratio, whereas responses to a PKC-activating phorbol ester were more sustained. In cells expressing D319N ERK2-GFP (D319N mutation impairs docking-domain-dependent binding to DUSPs), GnRH caused more sustained increases in ppERK2 and nuclear to cytoplasmic ERK2-GFP ratio and also had more pronounced effects on Egr-1 luciferase (a transcriptional reporter for ERK activation). Cycloheximide caused more sustained effects of GnRH and phorbol ester on ppERK, suggesting termination by nuclear-inducible DUSPs. GnRH also increased expression of nuclear-inducible DUSP1 and -4, but their knockdown did not alter GnRH-mediated ERK signaling. Screening a short inhibitory RNA library targeting 16 DUSPs (nuclear-inducible DUSPs, cytoplasmic ERK MAPK phosphatases, c-Jun N-terminal kinase/p38 MAPK phosphatases, and atypical DUSPs) revealed GnRH effects to be influenced by DUSPs 5, 9, 10, 16, and 3 (i.e. by each DUSP class). Thus, GnRH-mediated ERK responses (like PKC-mediated ERK responses) are dependent on protein neosynthesis and docking-domain-dependent binding, but for GnRH activation (unlike PKC activation), this does not reflect dependence on nuclear-inducible DUSPs. Termination of these GnRH effects is apparently dependent upon a preexisting rapid turnover protein.

Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility.

Background: Ttll5/STAMP is a multifunctional protein in cells with unknown activity in animals. Results: Targeted disruption of the Ttll5/Stamp gene in mice causes male infertility with reduced α-tubulin polyglutamylation and axoneme disruption in sperm. Conclusion: Ttll5/Stamp deficiency differs from previously described defects in sperm maturation and function. Significance: Ttll5/Stamp is a new gene involved in sperm maturation that may be relevant for human fertility. TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamptm/tm) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamptm/tm sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamptm/tm males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

Decoding GnRH neurohormone pulse frequency by convergent signalling modules

Gonadotropin-releasing hormone (GnRH) mediates control of reproduction. It is secreted in pulses and acts via intracellular effectors to activate gonadotrophin secretion and gene expression. Sub-maximal GnRH pulse frequency can elicit maximal responses, yielding bell-shaped frequency–response curves characteristic of genuine frequency decoders. GnRH frequency decoding is therapeutically important (pulsatile GnRH can drive ovulation in assisted reproduction whereas sustained activation can treat breast and prostate cancers), but the mechanisms are unknown. Here, we consider the possibility that it is due to convergence of distinct pulsatile signals at the transcriptome. We develop a model that mirrors wet-laboratory data for activation and nuclear translocation of GnRH effectors (extracellular signal regulated kinase and nuclear factors of activated T-cells) and incorporates transcription. The model predicts genuine frequency decoding when two transcription factors (TFs) converge at a cooperative gate, and shows how optimal pulse frequency could reflect TF activation kinetics and affinities. Importantly, this behaviour is revealed as an emergent feature of the network, rather than an intrinsic feature of a given protein or pathway, and since such network topology is extremely common, may well be widespread in biological systems.

Trafficking and signalling of gonadotrophin-releasing hormone receptors: an automated imaging approach

Gonadotrophin‐releasing hormone (GnRH) is a neuropeptide that mediates central control of reproduction by stimulating gonadotrophin secretion from the pituitary. It acts via 7 transmembrane region (7TM) receptors that lack C‐terminal tails, regions that for many 7TM receptors, are necessary for agonist‐induced phosphorylation and arrestin binding as well as arrestin‐dependent desensitization, internalization and signalling. Recent work has revealed that human GnRH receptors (GnRHR) are poorly expressed at the cell surface. This apparently reflects inefficient exit from the endoplasmic reticulum, which is thought to be increased by pharmacological chaperones (non‐peptide GnRHR antagonists that increase cell surface GnRHR expression) or reduced by point mutations that further impair GnRHR trafficking and thereby cause infertility. Here, we review recent work in this field, with emphasis on the use of semi‐automated imaging to interrogate compartmentalization and trafficking of these unique 7TM receptors.

Plasma membrane expression of gonadotropin-releasing hormone receptors: regulation by peptide and nonpeptide antagonists

Gonadotropin-releasing hormone acts via cell surface receptors but most human (h) GnRH receptors (GnRHRs) are intracellular. A membrane-permeant nonpeptide antagonist [(2S)-2-[5-[2-(2-axabicyclo[2.2.2]oct-2-yl)-1,1-dimethy-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine (IN3)] increases hGnRHR expression at the surface, apparently by facilitating its exit from the endoplasmic reticulum. Here we have quantified GnRHR by automated imaging in HeLa cells transduced with adenovirus expressing hemagglutinin-tagged GnRHR. Consistent with an intracellular site of action, IN3 increases cell surface hGnRHR, and this effect is not blocked or mimicked by membrane-impermeant peptide antagonists [Ac-D2Nal-D4Cpa-D3Pal-Ser-Tyr-d-Cit-Leu-Arg-Pro-d-Ala-NH(2) (cetrorelix) and antide]. However, when the C-terminal tail of a Xenopus (X) GnRHR was added (h.XGnRHR) to increase expression, both peptides further increased cell surface GnRHR. Cetrorelix also synergized with IN3 to increase expression of hGnRHR and a G-protein coupling-deficient mutant (A261K-hGnRHR). Cetrorelix also increased cell surface expression of hGnRHR, h.XGnRHR, and mouse GnRHR in gonadotrope-lineage LbetaT2 cells, and in HeLa cells it slowed h.XGnRHR internalization (measured by receptor-mediated antihemagglutinin uptake). Thus cetrorelix has effects other than GnRHR blockade; it acts as an inverse agonist in internalization assays, supporting the potential importance of ligand-biased efficacy at GnRHR. We also developed an imaging assay for GnRH function based on Ca(2+)-dependent nuclear translocation of a nuclear factor of activated T cells reporter. Using this in HeLa and LbetaT2 cells, IN3 and cetrorelix behaved as competitive antagonists when coincubated with GnRH, and long-term pretreatment (16 h) with IN3 reduced its effectiveness as an inhibitor whereas pretreatment with cetrorelix increased its inhibitory effect. This distinction between peptide and nonpeptide antagonists may prove important for therapeutic applications of GnRH antagonists.

Using automated imaging to interrogate gonadotrophin-releasing hormone receptor trafficking and function

Gonadotrophin-releasing hormone (GnRH) acts via seven transmembrane receptors on gonadotrophs to stimulate gonadotrophin synthesis and secretion, and thereby mediates central control of reproduction. Type I mammalian GnRHR are unique, in that they lack C-terminal tails. This is thought to underlie their resistance to rapid homologous desensitisation as well as their slow rate of internalisation and inability to provoke G-protein-independent (arrestin-mediated) signalling. More recently it has been discovered that the vast majority of human GnRHR are actually intracellular, in spite of the fact that they are activated at the cell surface by a membrane impermeant peptide hormone. This apparently reflects inefficient exit from the endoplasmic reticulum and again, the absence of the C-tail likely contributes to their intracellular localisation. This review is intended to cover some of these novel aspects of GnRHR biology, focusing on ways that we have used automated fluorescence microscopy (high content imaging) to explore GnRHR localisation and trafficking as well as spatial and temporal aspects of GnRH signalling via the Ca2+/calmodulin/calcineurin/NFAT and Raf/MEK/ERK pathways.