Gonadotropin-Releasing Hormone- Induced Rise in Cytosolic Free Ca<sup>2+</sup>Levels: Mobilization of Cellular and Extracellular Ca<sup>2+</sup>Pools and Relationship to Gonadotropin Secretion

Naor, Zvi; Capponi, Alessandro M.; Rossier, Michel F.; Ayalon, D.; Limor, Rona

doi:10.1210/mend-2-6-512

Cited by 77 publications

(41 citation statements)

References 31 publications

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“…In exploring this, we have found that sustained stimulation of T3-1 cells with GnRH causes a pronounced desensitisation of Ca 2+ responses (McArdle et al 1995(McArdle et al , 1996. In control cells, GnRH causes a spike-plateau type increase in the cytosolic [Ca 2+ ] I concentration in which the spike phase reflects IP 3 -mediated mobilisation of Ca 2+ from intracellular stores and the plateau phase reflects entry of Ca 2+ across the plasma membrane (McArdle et al 1992), as previously reported for primary cultures of rat pituitary cells (Naor et al 1988). Focusing on the mobilisation of intracellular Ca 2+ , we observed a relatively rapid desensitisation of GnRH-stimulated Ca 2+ mobilisation (Fig.…”

Section: Desensitisation Downstream Of the Gnrh-rsupporting

confidence: 85%

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

McArdle¹,

Franklin²,

Green³

et al. 2002

Journal of Endocrinology

160

103

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Sustained stimulation of G-protein-coupled receptors (GPCRs) typically causes receptor desensitisation, which is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of -arrestin not only prevents the receptor from activating its G protein (causing desensitisation), but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogenactivated protein kinase (MAPK) cascades. GnRH acts via phospholipase C (PLC)-coupled GPCRs on pituitary gonadotrophs to stimulate a Ca 2+ -mediated increase in gonadotrophin secretion. The type I GnRH receptors (GnRH-Rs), found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind -arrestin; they are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation, with concomitant receptor phosphorylation (within the C-terminal tails) or binding of -arrestin, or both. The association with -arrestin may also be important for regulation of dynamin, a GTPase that controls separation of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-Rs in Hela cells conditionally expressing a dominant negative mutant of dynamin (K44A), we have found that blockade of dynamindependent endocytosis inhibits internalisation of type II (xenopus) GnRH-Rs but not type I (human) GnRH-Rs. In these cells, blockade of dynamin-dependent internalisation also inhibited GnRH-R-mediated MAPK activation, but this effect was not receptor specific and therefore not dependent upon dynamin-regulated GnRH-R internalisation. Although type I GnRH-Rs do not desensitise, sustained activation of GnRH-Rs causes desensitisation of gonadotrophin secretion, and we have found that GnRH can cause down-regulation of inositol (1,4,5) trisphosphate receptors and desensitisation of Ca 2+ mobilisation in pituitary cells. The atypical resistance of the GnRH-R to desensitisation may underlie its atypical efficiency at provoking this downstream adaptive response. GnRH-Rs are also expressed in several extrapituitary sites, and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R-expressing adenovirus facilitated expression of high-affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells, and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a nondesensitising receptor. Thus it appears that the mammalian GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-Rs in pituitary and extrapituitary sites.

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Section: Desensitisation Downstream Of the Gnrh-rsupporting

confidence: 85%

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

McArdle¹,

Franklin²,

Green³

et al. 2002

Journal of Endocrinology

160

103

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“…The results imply that digitonin permeabilization is accompanied by loss of exocytotic components in addition to PKC. Furthermore, GnRH stimulation of gonadotropin secretion is most likely mediated by PKC (27)(28)(29)(30)(31)(32) and by other messenger molecules such as Ca2" and arachidonic acid and/or its metabolites as discussed elsewhere (33)(34)(35)(36)(37). However, since the exocytotic response elicited by PMA in normal cells ( Fig.…”

Section: Discussionmentioning

confidence: 92%

Induction of exocytosis in permeabilized pituitary cells by alpha- and beta-type protein kinase C.

Naor

Dan-Cohen

Hermon

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Protein kinase C is now recognized to comprise a family of closely related subspecies (PKCs). When cultured rat pituitary cells were permeabilized by digitonin for 5 min in the absence of Ca2+, endogenous PKC activity was decreased by 72%. PKC depletion was also achieved by prior treatment (24 hr) with high concentrations of phorbol 12-myristate 13-acetate (PMA). When purified activated brain PKCs were added for 30 min to PMA-pretreated, digitoninpermeabilized cells, only a-and fi-but not y-type PKC stimulated luteinizing hormone release. Since PKC was implicated as a mediator of gonadotropin secretion, gonadotropinreleasing hormone might utilize a-and fl-type PKCs for stimulation of gonadotropin secretion; a-and fl-type PKCs might participate also in other exocytotic responses in diverse biological systems in which PKC was implicated.Protein kinase C (PKC), which has been regarded as a single enzymatic activity, is now recognized as a family of closely related subspecies (PKCs; were cultured for 4 days in medium 199 containing 5% horse serum, penicillin at 100 units/ml, and streptomycin at 100 ,ug/ml, with or without 1 ,uM PMA for the last 24 or 48 hr of the culture period. Cells were then washed five times with cold phosphatebuffered saline and transferred (107 per tube) by rubber policeman to tubes containing 1 ml of homogenization buffer (20 mM Tris HCl, pH 7.5/0.25 M sucrose/10 mM EGTA/2 mM EDTA/1 mM phenylmethylsulfonyl fluoride/20 ,ug of leupeptin per ml). The cells were homogenized, and the homogenate was sonicated for 30 s at 4°C and incubated with 0.5% Triton X-100 for 30 min at 4°C to release the membraneassociated PKC. The homogenate was then centrifuged for 10 min at 100,000 x g (Beckman Airfuge, 207 kPa), and the supernatant was applied to a minicolumn of DE-52 cellulose equilibrated with 20 mM Tris HCI (pH 7.5) containing 0.5 mM EGTA, 0.5 mM EDTA, and 10 mM 2-mercaptoethanol (buffer A). The column was washed with 4 volumes of buffer A and then with 4 volumes of buffer A containing 20 mM NaCl. PKC was eluted with 4 volumes (=400 ,ul) of buffer A containing 120 mM NaCl. Samples of 20 ,ul (2 ,tg of protein) were subjected to the assay of PKC activity as described by Kikkawa et al. (15). The reaction mixture (0.25 ml) contained 5 ,umol ofTris HCl (pH 7.5), 1.25 ,mol of magnesium acetate, 50 ,g of histone (type III-S), 2.5 nmol of [y-32P]ATP (5-15 x 104 cpm/nmol), 8 ,ug of phosphatidylserine per ml, 0.8 ,ug of diolein per ml, 0.3 mM Ca2+, and the enzyme preparation.Basal activity was measured in the presence of 0.3 mM Ca2+ and was subtracted from the total activity. The assay was carried out for 20 min at 30°C. The reaction was stopped by the addition of 25% trichloroacetic acid; the acid-precipitate was collected on membrane filters (0.45 ,um) tTo whom reprint requests should be addressed. 4501The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this...

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“…receptor results in IP3-mediated increases in intracellular calcium which have been shown to be the primary signal for exocytosis in ␣T3-1 cells and primary gonadotropes (7)(8)(9). Calcium influx through the plasma membrane is thought to play a role in maintaining intracellular calcium stores.…”

Section: Gnrh-induced Activation Of Jnk Is Not Inhibited By Acute Chementioning

confidence: 99%

“…Extracellular calcium enters the cell through VGCCs in the plasma membrane while IP3 releases calcium from intracellular stores. The IP3-released calcium has been shown to be the critical signal required for secretion of the gonadotropic hormones, luteinizing hormone, and follicle-stimulating hormone (7,8). Influx of calcium through L-and T-type VGCCs is initiated independently from the IP3-mediated signal, but calcium influx through the plasma membrane is ultimately required for replenishment of intracellular stores (9).…”

mentioning

confidence: 99%

Divergent Signaling Pathways Requiring Discrete Calcium Signals Mediate Concurrent Activation of Two Mitogen-activated Protein Kinases by Gonadotropin-releasing Hormone

Mulvaney

Roberson

2000

Journal of Biological Chemistry

100

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Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor-and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in ␣T3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal ␣T3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.

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Gonadotropin-Releasing Hormone- Induced Rise in Cytosolic Free Ca²⁺Levels: Mobilization of Cellular and Extracellular Ca²⁺Pools and Relationship to Gonadotropin Secretion

Cited by 77 publications

References 31 publications

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

Induction of exocytosis in permeabilized pituitary cells by alpha- and beta-type protein kinase C.

Divergent Signaling Pathways Requiring Discrete Calcium Signals Mediate Concurrent Activation of Two Mitogen-activated Protein Kinases by Gonadotropin-releasing Hormone

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Gonadotropin-Releasing Hormone- Induced Rise in Cytosolic Free Ca2+Levels: Mobilization of Cellular and Extracellular Ca2+Pools and Relationship to Gonadotropin Secretion

Cited by 77 publications

References 31 publications

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

Induction of exocytosis in permeabilized pituitary cells by alpha- and beta-type protein kinase C.

Divergent Signaling Pathways Requiring Discrete Calcium Signals Mediate Concurrent Activation of Two Mitogen-activated Protein Kinases by Gonadotropin-releasing Hormone

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Gonadotropin-Releasing Hormone- Induced Rise in Cytosolic Free Ca²⁺Levels: Mobilization of Cellular and Extracellular Ca²⁺Pools and Relationship to Gonadotropin Secretion