Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies in several neuronal lineages suggest that extrinsic factors such as polypeptide growth factors regulate various stages of neuronal development, from initial commitment of multipotent progenitors to induction of specific gene expression that is characteristic of terminal neuronal differentiation. In the present study, immortalized hypothalamic neurons of the GT1-1 lineage were used to analyze proliferative, as well as morphological and molecular differentiation actions of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor-I (IGF-I). These effects were compared with those induced by specific activators of protein kinase A and C pathways, which potently inhibited cell proliferation and gonadotropin-releasing hormone (GnRH) gene expression, but stimulated morphological neuronal maturation as determined by the length and number of neurite outgrowth. bFGF exerted a broad spectrum of stimulatory effects, increasing the rate of proliferation measured both by the incorporation of 3H-thymidine and by cell number, and parameters of terminal differentiation, such as neurite outgrowth and induction of gene expression. bFGF stimulated the expression of the hybrid transgene-containing portions of the rat GnRH promoter. In contrast, EGF, TGF-alpha, and IGF-I inhibited cell proliferation, while having subtle effects on neurite outgrowth. Thus, GT1-1 cells appear to be differentially responsive to distinct neurotrophic factors, providing a model for studying the specific effects of neurotrophic factors on functional differentiation, migration, and connectivity of hypothalamic neurons.
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PMID:Differential effects of basic fibroblast growth factor, epidermal growth factor, transforming growth factor-alpha, and insulin-like growth factor-I on a hypothalamic gonadotropin-releasing hormone neuronal cell line. 933 61

We showed previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases glycoprotein hormone alpha-subunit gene expression and secretion in alphaT3-1 cells. We have now used 5'-flanking deletion and clustered point mutations of the mouse alpha-subunit promoter fused to the luciferase (LUC) reporter gene in transient transfection assays to further characterize the cell signaling pathways and sequences involved in responsiveness to PACAP. PACAP stimulated LUC activity at a lower concentration than VIP, supporting the notion that PACAP acts through type 1 receptors. The effect of PACAP on LUC activity was observed by 2 h, peaked at 4-12 h, and persisted until at least 20 h. alphaT3-1 cells were transfected with mouse alpha-LUC constructs truncated at -507, -424, -288, -205, -146, and -133, and treated with PACAP, a cell-permeable cAMP analog (8Br-cAMP), phorbol myristate acetate (PMA), or control medium. Transcriptional activation by PACAP was highest with the -288 and -205 mouse alpha-LUC vectors (7-8-fold stimulation) and decreased significantly with truncation of the 5'-flanking region to -146 or -133. The pattern of alpha-subunit stimulation by cAMP closely paralleled that of PACAP. With PMA, stepwise decrements in LUC activity were observed between -507 and -424 and, especially, -424 and -288, and there was no further loss of activity with deletion to -205, -146, or -133. Clustered point mutations in the pituitary glycoprotein hormone basal element (-337 to -330) or the gonadotropin-releasing hormone response element (GnRH-RE)(-406 to -399) of the -507 to +46 mouse alpha-promoter significantly (P < 0.05) increased and decreased, respectively, PACAP's effect on transcriptional activity. These results indicate that there are several regions of the mouse alpha-subunit promoter that mediate responsiveness to PACAP. The co-localization of PACAP and cAMP responsiveness as well as the results of studies involving specific inhibitors of protein kinase A (H-89) or protein kinase C (PKC) (bisindolylmaleimide) suggests that the action of PACAP on alpha-subunit transcription is mediated primarily by the protein kinase A (PKA) pathway.
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PMID:Transcriptional regulation of the glycoprotein hormone alpha-subunit gene by pituitary adenylate cyclase-activating polypeptide (PACAP) in alphaT3-1 cells. 960 11

The present study examined protein kinase A (PKA) and protein kinase C (PKC) involvement in the maintenance of cellular tolerance to mu opioid receptor agonists resulting from chronic opiate exposure in neurosecretory cells of the hypothalamic arcuate nucleus (ARC). The possibility that the diminution of mu opioid receptor/effector coupling produced by acute 17beta-estradiol or chronic opiate exposures is mediated by a common kinase pathway also was investigated. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized female guinea pigs. The mu opioid receptor agonist D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin (DAMGO) produced dose-dependent hyperpolarizations of ARC neurons. Chronic morphine treatment for 4 days reduced DAMGO potency 2.5-fold with no change in the maximal response. This effect was mimicked by a 20-min bath application of the PKA activator cAMP, Sp-isomer, or the PKC activator phorbol-12,13-dibutyrate. A 30-min bath application of the broad-spectrum protein kinase inhibitor staurosporine completely abolished the reduced DAMGO potency seen in morphine-tolerant neurosecretory cells, including those immunopositive for gonadotropin-releasing hormone. The effect of staurosporine was mimicked by the PKA inhibitor cAMP, Rp-isomer, but not by the PKC inhibitor calphostin C. Finally, a 20-min bath application of 17beta-estradiol did not further reduce DAMGO potency in morphine-tolerant ARC neurons. Therefore, increased PKA activity maintains cellular tolerance to mu opioid receptor agonists in ARC neurosecretory cells caused by chronic morphine treatment. Furthermore, acute 17beta-estradiol and chronic opiate treatments attenuate mu opioid receptor-mediated responses via a common PKA pathway.
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PMID:Protein kinase A maintains cellular tolerance to mu opioid receptor agonists in hypothalamic neurosecretory cells with chronic morphine treatment: convergence on a common pathway with estrogen in modulating mu opioid receptor/effector coupling. 961 32

Under control incubation conditions, gonadotropin-releasing hormone (GnRH) binds only a fraction of its receptors in rat-cultivated pituitary cells. Unmasking of the remaining receptors, which have been termed 'cryptic', requires drug- or peptide-induced protein kinase activation. Spontaneous masking however is not observed on pituitary cells sampled from castrated male rats, suggesting the presence of an intrinsic unmasking factor. Many endogenous factors could theoretically account for the effect. Here we attempted to identify the factor involved by taking advantage of their differential dependency upon second messengers and transduction cascades. Spontaneous unmasking of GnRH binding was found reversed by pertussis toxin (PTX), an inhibitor of alphai and alphao subunits of heterotrimeric G proteins, and by U73122, a phospholipase C (PLC) inhibitor. In contrast, desensitization of protein kinase C (PKC) or inhibition of tyrosine kinase by herbimycin were ineffective. Among endogenous pituitary factors able to unmask GnRH receptors in pituitary cells from normal male rats, as EGF, NPY or opiate peptides, only the latter were found to correspond to this transduction profile. In an attempt to characterize the pharmacology of opiate effects, naloxone (10 microM), a poorly selective opiate antagonist, restored masking of GnRH binding in cells from castrates. Only the delta antagonist naltrindole (1 microM) was able to mimick the action of naloxone. Conversely, when tested on cells from intact animals, morphine (10 microM), as well as dslet (1 microM) and met-ENK (10 nM), preferential delta agonists, but not dago and beta-endorphin or U50488 H and dynorphin, respectively micro and kappa agonists, were able to suppress masking. Among opioid peptides endogenous to the pituitary, only met-ENK was able to unmask cryptic receptors, an effect antagonized by naltrindole. We conclude that an opiate delta receptor subtype is endogenously activated in the pituitary of castrated male rats to prevent masking of GnRH binding.
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PMID:Delta opiate receptors account for the castration-induced unmasking of gonadotropin-releasing hormone binding sites in the rat pituitary. 987 2

Although gonadotropin-releasing hormone agonists (GnRHa) have been used in the therapy of the endocrine-dependent cancers, their biological mechanism remained obscure. We have studied the roles of mitogen-activated protein kinase family in the antiproliferative effect of GnRHa on the Caov-3 human ovarian cancer cell line. Reverse transcription-PCR assays confirmed mRNA for GnRH receptor in Caov-3 cells. In the presence of 1 microM GnRHa, the proliferation of cells was significantly reduced to 76% of controls after 24 h, and the effect was sustained up to 4 days. Although GnRHa had no effect on the activation of the Jun N-terminal kinase (JNK), treatment of Caov-3 cells with GnRHa activated extracellular signal-regulated protein kinase (ERK), and its effect was more than that induced by GnRH. Activation of ERK by GnRHa occurred within 5 min, with the maximum occurring at 3 h and sustained until 24 h. GnRHa also activated ERK kinase (mitogen-activated protein/ERK kinase) and resulted in an increase in phosphorylation of son of sevenless (Sos), and Shc. Furthermore, we examined the mechanism by which GnRHa induced ERK activation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, blocked the GnRHa-induced ERK activation. Phorbol 12-myristate 13-acetate (PMA) also induced the ERK activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of ERK by GnRHa. Elimination of extracellular Ca2+ by EGTA also did not abolish the activation of ERK by GnRHa. To examine the role of ERK cascade in the antiproliferative effect of GnRHa, PD98059, an inhibitor of mitogen-activated protein/ERK kinase, was used. This inhibitor canceled the antiproliferative effect of GnRHa and apparently reversed the GnRH-induced dephosphorylation of the retinoblastoma protein, the hyperphosphorylation of which is a hallmark of G1-S transition in the cell cycle. These results provide evidence that GnRHa stimulation of ERK activity may be mediated by Gbetagamma protein, not by PMA-sensitive protein kinase C nor extracellular Ca2+ in the Caov-3 human ovarian cancer cell line, suggesting that this cascade may play an important role in the antiproliferative effect of GnRHa.
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PMID:Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line. 1053 88

Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles.
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PMID:Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells. 1058 15

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.
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PMID:Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells. 1067 47

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbetagamma, and beta-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.
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PMID:Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone. 2855 Jan 41

Normal mammalian sexual maturation and reproductive functions require the integration and precise coordination of hormones at the hypothalamic, pituitary, and gonadal levels. Hypothalamic gonadotropin-releasing hormone (GnRH) is a key regulator in this system; after binding to its receptor (GnRHR), it stimulates de novo synthesis and release of gonadotropins in anterior pituitary gonadotropes. Since the isolation of the GnRHR cDNA, the expression of GnRHR mRNA has been detected not only in the pituitary, but also in extrapituitary tissues, including the ovary and placenta. It has been shown that change in GnRHR mRNA is one of the mechanisms for regulating the expression of the GnRHR. To help understand the molecular mechanism(s) involved in transcriptional regulation of the GnRHR gene, the 5' flanking region of the GnRHR gene has recently been isolated. Initial characterization studies have identified several DNA regions in the GnRHR 5' flanking region which are responsible for both basal expression and GnRH-mediated homologous regulation of this gene in pituitary cells. The mammalian GnRHR lacks a C-terminus and possesses a relatively short third intracellular loop; both features are important in desensitization of many others G-protein coupled receptors (GPCRs), Homologous desensitization of GnRHR has been shown to be regulated by various serine-threonine protein kinases including protein kinase A (PKA) and protein kinase C (PKC), as well as by G-protein coupled receptor kinases (GRKs). Furthermore, GnRHR was demonstrated to couple with multiple G proteins (Gq/11, Gs, and Gi), and to activate cascades that involved the PKC, PKA, and mitogen-activator protein kinases. These results suggest the diversity of GnRHR-G protein coupling and signal transduction systems. The identification of second form of GnRH (GnRH-II) in mammals adds to the complexity of the GnRH-GnRHR system. This review summaries our recent progress in understanding the regulation of GnRHR gene expression and the GnRHR signal transduction pathways.
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PMID:The expression, regulation and signal transduction pathways of the mammalian gonadotropin-releasing hormone receptor. 1114 80

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.
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PMID:cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel. 1137 17


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