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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Binding sites for LH/hCG are found in the uterus of several species, including humans. In cattle and pigs, the LH receptor, its mRNA and LH receptor protein are present in the uterus throughout the oestrous cycle, and maximum expression occurs at the luteal phase. GnRH receptor is also expressed maximally in the human endometrium at the luteal phase. LH activates both the adenylate cyclase and phospholipase C pathways and increases the concentrations of cyclooxygenase and its products. Activation of LH receptors in the endometrium is associated with PGF production. In contrast, bovine uterine vein LH receptor mRNA and LH receptor concentrations are greatest during pro-oestrus-oestrus and LH increases the production of both PGE and PGF. FSH receptor and its mRNA are present in the bovine cervix and the concentrations are greatest at the time of the FSH peak value in the blood, indicating a physiological role for FSH in the relaxation and opening of the cervix. The presence of gonadotrophin and releasing factor receptors with a dynamic pattern in the endometrium, myometrium, oviduct and cervix of different species provides evidence that gonadotrophins and GnRH play a substantial role as molecular autocrine-paracrine regulators of the oestrous cycle and implantation.
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PMID:Actions of gonadotrophins on the uterus. 1137 69

The GnRH-R belongs to the superfamily of heptahelical GPCRs. A three-dimensional model of GnRH binding to its receptor predicted that Trp3 was the most deeply buried residue, potentially allowing it to interact with both Trp279, a highly conserved residue in the TMH 6 of GPCRs, and Phe310, present essentially in TMH 7 of GnRH-Rs. Replacement of Phe310 with Leu, the most common positional residue in GPCRs, induced a slightly decreased Bmax (1.6-fold) and affinity (3.8-fold); in addition, IP production was completely abolished. Similarly, replacement of Trp279 with Ser depressed the Bmax by 5.2-fold, the affinity by 2.3-fold, and totally abrogated IP production. The effect of the double mutation was not additive on binding, since the Bmax was reduced to the level of the Phe310Leu mutant, although the Kd was restored to a value not significantly different from that of the wild-type. The double mutant was also unable to induce IP production. Unexpectedly, no influence of any single or double substitution was noted on receptor internalization. These data provide evidence for the crucial role of Phe310, possibly in conjunction with Trp279, on GnRH transduction and suggest that the conformation for phospholipase C activation may not be required for GnRH-R internalization.
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PMID:Critical implication of transmembrane Phe310, possibly in conjunction with Trp279, in the rat gonadotropin-releasing hormone receptor activation. 1143 5

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.
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PMID:Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling. 1149 5

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 beta-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 mitogen-activated 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 beta-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 beta-arrestin, or both. The association with beta-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 dynamin-dependent 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 non-desensitising 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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PMID:Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors. 1192 79

Mutations of the GnRH receptor have been recognized as a cause of familial gonadotropin deficiency. We here identify and functionally characterize a novel human GnRH receptor variant bearing an Ala(171)Thr substitution located at transmembrane helix 4 (TMH4). The affected kindred displays severe hypogonadotropic hypogonadism. After in vitro expression in human embryonic kidney 293T cells, the Ala(171)Thr mutant GnRH receptor exhibited a lack of phospholipase C activity in signal transduction. Specific receptor binding of (125)I-labeled GnRH ligand was undetectable in Ala(171)Thr GnRH receptor-transfected cells. Molecular modeling and dynamic simulation of the Ala(171)Thr GnRH receptor suggests the introduction of a stable hydrogen bond between residue Thr(171) and Tyr(119) side-chains at a distance of 2 A. Although spatially distant from the GnRH ligand-binding site, this hydrogen bond impedes conformational mobility of the TMH3 and TMH4 domains required for sequential ligand binding and receptor activation, thus stabilizing the GnRH receptor in its inactive conformation. Receptor structure modeling and functional data provide a comprehensive molecular view of how mutation Ala(171)Thr causes a complete loss of GnRH receptor function.
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PMID:Mutation Ala(171)Thr stabilizes the gonadotropin-releasing hormone receptor in its inactive conformation, causing familial hypogonadotropic hypogonadism. 1267 86

Activation of classical G protein-coupled receptors (GPCRs) like the mammalian gonadotropin-releasing hormone receptor (GnRHR) typically stimulates heterotrimeric G protein molecules that subsequently activate downstream effectors. Receptor activation of heterotrimeric G protein pathways primarily controls intermediary cell metabolism by elevation or diminution of soluble cytoplasmic second messenger molecules. We have demonstrated here that stimulation of the GnRHR also results in a dramatic change in both cell adhesion and superstructural morphology. Gonadotropin-releasing hormone (GnRH) receptor activation rapidly increases the capacity of HEK293 cells expressing the GnRHR to remain matrix-adherent in the face of fluid insults. Coinciding with this profound elevation in matrix adherence, we demonstrated a GnRH-induced alteration in both cell morphology and the de novo generation of polymerized actin structures. GnRH induction of cytoskeletal remodeling was correlated with significant increases in the tyrosine phosphorylation status of a series of cytoskeletal associated proteins, e.g. focal adhesion kinase (FAK), c-Src, and microtubule-associated protein kinase (MAPK or ERK1/2). The activation of the distal downstream effector ERK1/2 was demonstrated to be sensitive to the disrupters of cytoskeletal rearrangement, cytochalasin D and latrunculin B. In addition to the sensitivity of ERKs to cytoskeletal integrity, GnRH-induced FAK and c-Src kinase activation were sensitive to these agents and the fibronectin-integrin antagonistic RGDS peptide. Activation of ERK was dependent on its protein-protein assembly with FAK and c-Src at focal adhesion complexes. Induction of the cell remodeling event leading to this signaling complex assembly occurred primarily via GnRHR activation of the monomeric G protein Rac but not RhoA. These findings demonstrated a clear divergence of GnRHR signaling via the Rac monomeric G protein focal adhesion signaling complex assembly and cytoskeletal remodeling independent of the classical heterotrimeric G protein-controlled phospholipase C-beta pathway.
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PMID:Cytoskeletal reorganization dependence of signaling by the gonadotropin-releasing hormone receptor. 1455 94

Recently, we demonstrated that the mammalian type-I GnRH receptor (GnRHR) has a high preference for the phospholipase C/protein kinase C (PLC/PKC)-linked signaling pathway, whereas non-mammalian bullfrog (bf) GnRHRs couple to both adenylate cyclase/protein kinase A (AC/PKA)- and PLC/PKC-linked signaling pathways. In the pre-sent study, using AC/PKA-specific reporter (cAMP-responsive element-luciferase) and PLC/PKC-specific reporter (serum-responsive element-luciferase) systems, we attempted to identify the motif responsible for this difference. A deletion of the intracellular carboxyl-terminal tail (C tail) of bfGnRHR-1 remarkably decreased its ability to induce the AC/PKA-linked signaling pathway. Further dissection of the C tail indicated that an HFRK motif in the membrane-proximal sequence of bfGnRHR-1 C tail is a minimal requirement for the AC/PKA-linked signaling pathway as the addition of this motif to rat GnRHR or deletion of it from bfGnRHR-1 significantly affected the ability to induce the AC/PKA-linked signaling pathway. Deletion or addition of the HFRK motif, however, did not critically influence the PLC/PKC-linked signaling pathway. These results indicate that the HFRK motif in the membrane-proximal region confers the differential signal transduction pathways between mammalian and nonmammalian GnRHRs.
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PMID:Membrane-proximal region of the carboxyl terminus of the gonadotropin-releasing hormone receptor (GnRHR) confers differential signal transduction between mammalian and nonmammalian GnRHRs. 1556 46

Chronic GnRH treatment causes homologous desensitization by reducing GnRH receptor and Gq/11 expression and by down-regulating protein kinase C (PKC), cAMP, and calcium-dependent signaling. It also causes heterologous desensitization of other Gq-coupled receptors, but the mechanisms involved remain elusive. In this study, we investigated the effect of constitutive activation of Gq signaling on GnRH-induced signaling and LH secretion. We show that adenoviral expression of a constitutively active mutant Gq(Q209L) results in a state of GnRH resistance but does not alter GnRH receptor expression. We observed that Gq(Q209L) reduced expression of phospholipase C (PLC)beta1, a target of Gq in these cells, but not PLCbeta3 or PLCgamma1. Downstream of PLCbeta1, expression of novel PKC isoforms (delta and epsilon) was reduced. Adenoviral expression of a kinase-inactive, dominant-negative version of PKCdelta impaired GnRH activation of ERK, but not induction of c-Fos and LHbeta proteins, indicating that the novel PKCs signal to the ERK cascade. Despite reductions in PLCbeta1, calcium responses to GnRH were elevated in Gq(Q209L)-infected cells due to increased calcium influx through L-type calcium channels. Paradoxically, downstream calcium-dependent signaling and LH secretion were impaired. Taken together, these data demonstrate that prolonged activation of the Gq pathway desensitizes GnRH-induced signaling by selectively down-regulating the PLC-PKC-Ca2+ pathway, leading to reduced LHbeta synthesis and LH secretion.
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PMID:Constitutively active Gq impairs gonadotropin-releasing hormone-induced intracellular signaling and luteinizing hormone secretion in LbetaT2 cells. 1587 57

Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in alphaT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone alpha-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.
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PMID:Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin. 1589 Jun 71

Activation of seven-transmembrane region receptors typically causes their phosphorylation with consequent arrestin binding and desensitization. Arrestins also act as scaffolds, mediating signaling to Raf and ERK and, for some receptors, inhibiting nuclear translocation of ERK. GnRH receptors (GnRHRs) act via Gq/11 to stimulate the phospholipase C/Ca2+/protein kinase C (PKC) cascade and the Raf/MEK/ERK cassette. Uniquely, type I mammalian GnRHRs lack the C-tails that are found in other seven-transmembrane region receptors (including nonmammalian GnRHRs) and are implicated in arrestin binding. Here we have compared ERK signaling by human GnRHRs (hGnRHRs) and Xenopus GnRHRs (XGnRHRs). In HeLa cells, XGnRHRs underwent rapid and arrestin-dependent internalization and caused arrestin/green fluorescent protein (GFP) translocation to the membrane and endosomes, whereas hGnRHRs did not. Internalized XGnRHRs were co-localized with arrestin-GFP, whereas hGnRHRs were not. Both receptors mediated transient ERK phosphorylation and nuclear translocation (revealed by immunohistochemistry or by imaging of co-transfected ERK2-GFP), and for both, ERK phosphorylation was reduced by PKC inhibition but not by inhibiting epidermal growth factor receptor autophosphorylation. In the presence of PKC inhibitor, Deltaarrestin-(319-418) blocked XGnRHR-mediated, but not hGnRHR-mediated, ERK phosphorylation. When receptor number was varied, hGnRHRs activated phospholipase C and ERK more efficiently than XGnRHRs but were less efficient at causing ERK2-GFP translocation. At high receptor number, XGnRHRs and hGnRHRs both caused ERK2-GFP translocation to the nucleus, but at low receptor number, XGnRHRs caused ERK2-GFP translocation, whereas hGnRHRs did not. Thus, experiments with XGnRHRs have revealed the first direct evidence of arrestin-mediated (probably G protein-independent) GnRHR signaling, whereas those with hGnRHRs imply that scaffolds other than arrestins can determine GnRHR effects on ERK compartmentalization.
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PMID:Arrestin-mediated ERK activation by gonadotropin-releasing hormone receptors: receptor-specific activation mechanisms and compartmentalization. 1631 13


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