Rapid Down-regulation of the Type I Inositol 1,4,5-Trisphosphate Receptor and Desensitization of Gonadotropin-releasing Hormone-mediated Ca2+ Responses in αT3–1 Gonadotropes

Willars, Gary B.; Royall, Jean E.; Nahorski, Stefan R.; El-Gehani, Faraj; Everest, Helen M.; McArdle, Craig A.

doi:10.1074/jbc.m008916200

Cited by 63 publications

(55 citation statements)

References 35 publications

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“…3) Fig. 3), the wild-type GnRHRs mediated the anticipated robust increase in [Ca 2+ ] i (see also Hislop et al 2001, Willars et al 2001) and a similar response was seen in cells transfected with the h.XtGnRHR construct. No such increase was seen in cells transfected with the h.XlGnRHR although this may simply reflect low expression of the receptor (results not shown).…”

Section: Resultsmentioning

confidence: 91%

Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization?

Hislop¹,

Caunt²,

Sedgley³

et al. 2005

Journal of Molecular Endocrinology

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Activation of seven-transmembrane receptors is typically followed by desensitization and arrestin-dependent internalization via vesicles that are pinched off by a dynamin collar. Arrestins also scaffold Src, which mediates dynamin-dependent internalization of 2-adrenergic receptors. Type I mammalian gonadotropin-releasing hormone receptors (GnRHRs) do not rapidly desensitize or internalize (characteristics attributed to their unique lack of C-terminal tails) whereas non-mammalian GnRHRs (that have C-terminal tails) are rapidly internalized and desensitized. Moreover, internalization of Xenopus (X) GnRHRs is dynamin-dependent whereas that of human (h) GnRHRs is not, raising the possibility that binding of arrestin to the C-terminal tails of GnRHRs targets them to the dynamin-dependent internalization pathway. To test this we have compared wild-type GnRHRs with chimeric receptors (XGnRHR C-terminal tail added to the hGnRHR alone (h.XtGnRHR) or with exchange of the third intracellular loops (h.Xl.XtGnRHR)). We show that adding the XGnRHR C-terminal tail facilitates arrestin-and dynamin-dependent internalization as well as arrestin/green fluorescent protein translocation, but Src (or mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibition does not slow internalization, and h.XtGnRHR internalization is slower than that of the hGnRHR. Moreover, arrestin expression increased XGnRHR internalization even when dynamin was inhibited and h.Xl.XtGnRHR underwent rapid arrestin-dependent internalization without signaling to G q/11 . Thus, although the C-terminal tail can direct GnRHRs for arrestin-and dynamin-dependent internalization, this effect is not dependent on Src activation and arrestin can also facilitate dynamin-independent internalization.

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Section: Resultsmentioning

confidence: 91%

Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization?

Hislop¹,

Caunt²,

Sedgley³

et al. 2005

Journal of Molecular Endocrinology

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“…As this occurs in the absence of receptor desensitisation and is not attributable to depletion of the intracellular Ca 2+ pool, it appears to reflect a reduction in the efficiency with which IP 3 mobilises Ca 2+ from this pool (McArdle et al 1996). This effect was associated with, and apparently dependent upon, a GnRH-stimulated reduction in the number of IP 3 receptors (IP 3 -Rs) (Willars et al 2001). Sustained activation of several PLC-activating GPCRs has been shown to cause IP 3 -R down-regulation, an effect attributed to the ligand-stimulated ubiquitinylation and consequent proteolysis of the activated IP 3 -R (Wojcikiewicz & Oberdorf 1996, Zhu & Wojcikiewicz 2000.…”

Section: Desensitisation Downstream Of the Gnrh-rmentioning

confidence: 97%

“…80% reduction within 20-30 min). Thus it appears that this receptor, which is atypically resistant to desensitisation, is atypically efficient at provoking this novel form of post-receptor desensitisation (Willars et al 2001). It is important to recognise, however, that the relevance of this effect to desensitisation of gonadotrophin secretion has not been established.…”

Section: Desensitisation Downstream Of the Gnrh-rmentioning

confidence: 99%

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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“…Therefore upon stimulation the Type I GnRH receptor chronically activates phospholipase C-␤. It has, however, been demonstrated that there is a regulatory process upon GnRH-mediated signaling at the level of inositol trisphosphate receptor activation and subsequent intracellular calcium mobilization (3). The protracted GnRH receptor stimulation causes a physical downregulation of the intracellular inositol trisphosphate receptors in the gonadotrope resulting in a curtailment upon any successive ligand-mediated intracellular Ca 2ϩ pulses.…”

mentioning

confidence: 99%

Gonadotropin-releasing Hormone-induced Activation of Diacylglycerol Kinase-ζ and Its Association with Active c-Src

Davidson

Pawson

Maturana

et al. 2004

Journal of Biological Chemistry

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Gonadotropin-releasing hormone (GnRH)-induced receptor activation has been demonstrated to entrain a wide variety of signaling modalities. Most signaling pathways are concerned with the control of serine, threonine, or tyrosine-protein kinases, however, in the current article we demonstrate that in both a model cell line and in gonadotropes, GnRH additionally mediates the activation of lipid-directed kinases. We have shown that there is a functional connection between proteintyrosine kinase modulation and lipid kinase activation. In HEK293 cells stably expressing the Type I mammalian GnRH receptor, we employed a proteomic approach to identify novel protein binding partners for GnRHactivated c-Src. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry we identified a GnRH-induced association between c-Src and the lipid kinase, diacylglycerol kinase-(DGK-). Using reciprocal co-immunoprecipitation we show that there is a significant elevation of the association between catalytically active c-Src with DGK-in both HEK293 cells and murine gonadotrope L␤T2 cells. Employing lipid kinase assays we have shown that the catalytic activity of DGK-is significantly heightened in both HEK293 and L␤T2 cells by GnRH. In addition, we demonstrate that the activation of DGK-exerts a functional role in the murine gonadotrope L␤T2 cell line. Elevated expression of DGK-resulted in a shortening of the time scale of ERK activation in these cells suggesting a potential role of endogenous DGK-in controlling the induction of LH␤ transcription by ERK1/2.Gonadotropin-releasing hormone (GnRH) 1 is the central regulator of the reproductive system and was first isolated as a decapeptide from mammalian hypothalami. GnRH released from the hypothalamus activates heptahelical G protein-coupled receptors (GPCRs) specific for GnRH on pituitary gonadotrope cells and mediates the secretion of the gonadotropins, luteinizing (LH) and follicle-stimulating hormone. This process is controlled, in part, by the elevation of intracellular calcium and the activation of protein kinase C (for review see Ref. 1). GnRH initiates both of these processes via the activation of phospholipase C-␤, which hydrolyzes phosphatidylinositol bisphosphate forming inositol trisphosphate and diacylglycerol (DAG). These two metabolites generate the intracellular signals required for gonadotropin secretion thus facilitating consistent reproductive function. Unlike most other GPCRs the Type I GnRH receptor does not possess an intracellular carboxyl terminus, which is typically involved in autoregulation of the receptors signaling activity (for review see Ref.2). Therefore upon stimulation the Type I GnRH receptor chronically activates phospholipase C-␤. It has, however, been demonstrated that there is a regulatory process upon GnRH-mediated signaling at the level of inositol trisphosphate receptor activation and subsequent intracellular calcium mobilization (3). The protracted GnRH receptor stimulation causes a physical downregulation of the intracellular...

show abstract

Rapid Down-regulation of the Type I Inositol 1,4,5-Trisphosphate Receptor and Desensitization of Gonadotropin-releasing Hormone-mediated Ca2+ Responses in αT3–1 Gonadotropes

Cited by 63 publications

References 35 publications

Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization?

Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization?

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors

Gonadotropin-releasing Hormone-induced Activation of Diacylglycerol Kinase-ζ and Its Association with Active c-Src

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