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)

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.
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PMID:The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein. 131 Sep 43

Xenopus oocytes that express mouse thyrotropin-releasing hormone receptors (TRH-Rs) after injection if RNA transcribed from TRH-R cDNA respond to THR by a depolarizing current. This response is transduced by activation of phosphoinositide-specific phospholipase C and utilizes an as yet unidentified endogenous guanine nucleotide-binding regulatory (G) protein(s). The alpha subunit of G11 and Gq have recently been shown to couple receptors to activation of phospholipase C. To determine whether there are functional differences between these proteins, we have co-expressed the TRH-R with either alpha 11 or alpha q. alpha 11 potentiated the response to TRH (by 61 +/- 16%), while alpha q inhibited the response (by 37 +/- 9%). The changes in amplitudes were accompanied by inverse changes in response latencies. These data show that alpha 11 and alpha q differentially modulate signal transduction in Xenopus oocytes.
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PMID:G alpha 11 and G alpha q guanine nucleotide regulatory proteins differentially modulate the response to thyrotropin-releasing hormone in Xenopus oocytes. 164 77

In GH4C1 rat pituitary cells, a GTP-binding protein appears to be involved in signal transduction between the TRH receptor and phospholipase C. In certain other cell types, another role for GTP has been reported, namely regulation of Ca2+ translocation from one intracellular pool to another. Using digitonin-permeabilized GH4C1 cells, we have investigated whether an analogous process occurs in pituitary cells. In permeabilized GH4C1 cells, TRH, inositol 1,4,5-trisphosphate (IP3), and nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and 5'-guanylyl imidodiphosphate each increased free Ca2+ concentration [( Ca2+]). Unlike several other systems, GTP did not increase [Ca2+]. Guanosine 5'-O-(2-thiodiphosphate) inhibited Ca2+ release induced by both TRH and GTP gamma S. Heparin abolished IP3-induced Ca2+ release but did not prevent Ca2+ release induced by TRH or GTP gamma S, suggesting a mechanism for their actions that did not depend solely on IP3 production. Neomycin inhibited GTP gamma S-induced Ca2+ release, but it did not prevent TRH- or IP3-induced Ca2+ release. In the absence of ATP, GTP gamma S did not elevate [Ca2+], although TRH and IP3 did, suggesting that ATP-dependent sequestration of Ca2+ was necessary for the action of GTP gamma S in this system, but not for TRH and IP3. Repeated additions of IP3 resulted in an attenuation of the response to IP3- GTP gamma S, which itself increased [Ca2+] after IP3 attenuation, restored the attenuated Ca2+ response to IP3. We conclude that, in permeabilized GH4C1 cells, GTP gamma S as well as TRH cause intracellular Ca2+ release; however, their mechanisms of action are, at least in part, distinct. Furthermore, the IP3-depletable Ca2+ pool can be refilled from a GTP gamma S-sensitive compartment via Ca2+ transport through the cytosol.
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PMID:Control of intracellular calcium redistribution by guanine nucleotides and inositol 1,4,5-trisphosphate in permeabilized GH4C1 cells. 190 95

The effect of decreasing the concentration of receptors for thyrotropin-releasing hormone (TRH) on the surface of cloned rat pituitary (GH3) cells on TRH-stimulated inositol trisphosphate (Ins-P3) formation was investigated. Incubation of cells with dibutyryl cAMP (Bt2cAMP) for 16 h caused a decrease in [3H] TRH binding to intact cells to a minimum level 37 +/- 9.1% of control. Scatchard analysis of the concentration dependency of [3H]TRH binding showed that the effect of Bt2cAMP was to lower the receptor concentration without affecting its affinity for TRH. Similar decreases in [3H]TRH binding were found in cells incubated with 8-bromo-cAMP, cholera toxin, and sodium butyrate and, as shown previously, with TRH. In cells incubated with 1 mM Bt2cAMP for 16 h, but not for 1 h, the maximum TRH-induced increase in Ins-P3 was inhibited to 25 +/- 3.2% of that in control cells. Inhibition of TRH-induced Ins-P3 formation was also observed in cells treated with 8-bromo-cAMP, cholera toxin, and sodium butyrate for 16 h, and with TRH for 48 h. Inhibition of TRH-induced Ins-P3 formation and lowering of TRH receptor concentration caused by Bt2cAMP occurred in parallel with increasing doses of Bt2cAMP; at 16 h of exposure, half-maximal effects occurred with 0.3 mM Bt2cAMP. The concentration dependency of TRH-induced Ins-P3 formation was the same in control and Bt2cAMP-treated cells; half-maximal effects occurred with 10 nM TRH. These data demonstrate that decreases in TRH receptor concentration caused by several agents that act via different mechanisms are associated with reduced stimulation of Ins-P3 formation and suggest that the TRH receptor is tightly coupled to stimulation of hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phospholipase C.
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PMID:Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells. 299 79

Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a phospholipase C to produce inositol trisphosphate (InsP3) and 1,2-diacylglycerol appears to be the initial step in signal transduction for a number of cell-surface interacting stimuli, including thyrotropin-releasing hormone (TRH). In suspensions of membranes isolated from rat pituitary (GH3) cells that were prelabeled to isotopic steady state with [3H]inositol and incubated with ATP, [3H] PtdIns(4,5)P2, and [3H]phosphatidylinositol 4-phosphate, the polyphosphoinositides, and [3H]InsP3 and [3H]inositol bisphosphate, the inositol polyphosphates, accumulated. TRH and GTP stimulated the accumulation of [3H]inositol polyphosphates in time- and concentration-dependent manners; half-maximal effects occurred with 10-30 nM TRH and with 3 microM GTP. A nonhydrolyzable analog of GTP also stimulated [3H] inositol polyphosphate accumulation. Moreover, when TRH and GTP were added together their effects were more than additive. Fixing the free Ca2+ concentration in the incubation buffer at 20 nM, a value below that present in the cytoplasm in vivo did not inhibit stimulation by TRH and GTP of [3H]inositol polyphosphate accumulation. ATP was necessary for basal and stimulated accumulation of [3H]inositol polyphosphates, and a nonhydrolyzable analog of ATP could not substitute for ATP. These data demonstrate that TRH and GTP act synergistically to stimulate the accumulation of InsP3 in suspensions of pituitary membranes and that ATP, most likely acting as substrate for polyphosphoinositide synthesis, was necessary for this effect. These findings suggest that a guanine nucleotide-binding regulatory protein is involved in coupling the TRH receptor to a phospholipase C that hydrolyzes PtdIns(4,5)P2.
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PMID:Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells. 300 61

In a crude membrane preparation of rat 7315c cells, GTP was found to enhance thyrotropin-releasing hormone- (TRH) stimulated inositol triphosphate (IP3) formation with a potency of 0.97 +/- 0.1 microM. TRH stimulation of IP3 formation was inhibited by high GDP concentrations. Neither nucleotide had any effect in the absence of TRH. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) stimulated IP3 formation in the absence of TRH; the apparent affinity of GTP gamma S was 0.16 +/- 0.05 microM. GTP blocked GTP gamma S stimulation of IP3 formation in a concentration-dependent manner. The apparent affinity of GTP for the site of action shared by GTP gamma S was calculated to be 0.98 +/- 0.3 microM. TRH was able to reverse inhibition of GTP gamma S-stimulated IP3 formation by GTP but could not reverse inhibition by GDP. A lag in the rate of IP3 formation in response to GTP gamma S was abolished by addition of TRH. These data support the proposal that activation of the TRH receptor enhances turnover of guanine nucleotides at the binding protein coupling the receptor to phospholipase C. In addition, GTP gamma S diminished high affinity [3H]Me-TRH binding. The potency of GTP gamma S at decreasing [3H]Me-TRH binding was 0.092 +/- 0.03 microM. GTP gamma S (0.1 microM) decreased the affinity of the TRH receptor for [3H]Me-TRH from 2 to 100 nM. Maximally effective concentrations of GTP gamma S, Gpp(NH)p, GTP, and GDP decreased specific [3H]Me-TRH binding by 80%. Pretreatment of cells with pertussis toxin (30 ng/ml for 24 h) failed to affect TRH receptor affinity or the potency or efficacy of GTP gamma S in diminishing [3H]Me-TRH binding, supporting the identification of Gp (a GTP-binding protein associated with phospholipase C and Ca2+-mobilizing receptors) as distinct from Gi (an inhibitory GTP-binding protein). In contrast to its lack of effect on TRH receptor binding, 3-h pertussis toxin treatment decreased agonist affinity of the mu-opiate receptor and abolished the ability of GTP gamma S to shift the affinity of the mu-opiate receptor for its agonist. The affinities calculated for GTP, GDP, GTP gamma S, and Gpp (NH)p for the G-protein regulating receptor affinity and IP3 formation are nearly identical for each guanine nucleotide tested, suggesting the same G-protein regulates both activities.
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PMID:Regulation of thyrotropin-releasing hormone receptor binding and phospholipase C activation by a single GTP-binding protein. 303 63

It is now established that a key step in the action of calcium-mobilizing agonists is stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). The latter substance acts as a second messenger, controlling the release of calcium from intracellular stores (see ref. 3 for review). The bifurcating nature of the signalling system is exemplified by the fact that the other product of PtdIns(4,5)P2 hydrolysis, 1,2-diacylglycerol, can alter cellular function by activating protein kinase C, the cellular target for several tumour-promoting agents such as the phorbol esters. In various tissues, including GH3 pituitary tumour cells, a synergistic interaction between calcium ions and protein kinase C underlies agonist-induced changes in cell activity. The data presented here suggest that when GH3 cells are stimulated by thyrotropin-releasing hormone (TRH), an agonist inducing PtdIns(4,5)P2 hydrolysis, the two limbs of the inositol lipid signalling system interact to control free cytosolic calcium levels [( Ca2+]i). At low levels of TRH receptor occupancy, [Ca2+]i increases rapidly, then declines relatively slowly. As receptor occupancy increases, the calcium signal becomes more short-lived due to the appearance of a second, inhibitory, component. This latter component, which is enhanced when [Ca2+]i is elevated by high potassium depolarization, is mimicked by active phorbol esters and by bacterial phospholipase C. It seems likely that protein kinase C subserves a negative feedback role in agonist-induced calcium mobilization.
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PMID:Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells. 392 49

TRH and GnRH receptors are each coupled to G proteins of the Gq/11 family. Activation of each of these receptors by their respective ligands results in the stimulation of phospholipase C activity, leading to calcium mobilization and protein kinase C activation. Thus, the effects of TRH and GnRH may be mediated through the same intracellular signal transduction pathway. To compare responses to TRH and GnRH directly within one cell type, we have stably transfected the rat pituitary GH3 lactotrope cell line, which expresses the endogenous TRH receptor, with an expression vector containing rat GnRH receptor cDNA. Transfected cells specifically bound GnRH with high affinity and responded to GnRH stimulation with an increase in PRL mRNA levels, analogous to their response to TRH stimulation. Stably transfected GH3 cells, which were then transiently transfected with luciferase reporter constructs containing either the PRL or the glycoprotein hormone alpha-subunit promoter, responded to either GnRH or TRH stimulation with an increase in luciferase activity in a time- and dose-dependent fashion. The stimulatory effects of maximally effective concentrations of TRH and GnRH were additive on PRL, but not alpha-subunit, gene expression. These data, coupled with evidence of cross-desensitization of alpha-subunit, but not PRL, promoter activity stimulation by TRH and GnRH, suggest that there may be differences in the signal transduction pathways activated by TRH and GnRH receptors in the regulation of PRL and alpha-subunit gene expression.
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PMID:Evidence that signalling pathways by which thyrotropin-releasing hormone and gonadotropin-releasing hormone act are both common and distinct. 752 98

TRH receptor-related signal transduction mechanism in the pituitary cells and the central nervous system was reviewed. In pituitary cells, TRH binds to its specific receptor on the cell membrane, followed by hydrolysis of inositol phospholipids by activation of phospholipase C leading to an increase in inositol 1,4,5-trisphosphates (IP3) and diacylglycerol (DG). IP3 mobilizes intracellular Ca2+, which activates Ca2+ and Calmodulin dependent protein kinase (Ca-CaM kinase) and DG activates protein kinase C (PKC). Both Ca-CaM kinase and PKC phosphorylates several proteins in the nucleus, plasma membranes, and cytosol resulting in cell responses including hormone secretion and gene expression. Protein dephosphorylation is also involved in TRH action in the pituitary. In the central nervous system, TRH possesses different intracellular signaling systems, which vary with brain regions.
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PMID:[TRH receptor-related signal transduction mechanism]. 819 62

Human embryonic kidney 293 cells that had been transfected to express the long isoform of the rat thyrotropin-releasing hormone (TRH) receptor (clone E2) were further transfected with a cDNA encoding the murine version of G11alpha. A clone was isolated (clone E2M11) that stably expressed murine as well as the endogenous human G11alpha. Subcellular fractionation demonstrated identical cellular distribution of the two species variants of this G protein. Sustained exposure of clone E2M11 cells to TRH resulted in substantial cellular redistribution and reduction in total cellular levels of G11alpha immunoreactivity. Fractions of both the exogenously introduced murine and endogenously expressed human isoforms of G11alpha were transferred from plasma membranes to low density membranes (detected as a shift from middle to low density regions on sucrose density gradients) and cytosol fractions. The plasma membrane redistribution to low density membrane was accompanied by a parallel redistribution of G protein beta subunits; however, there was no increase in beta subunits in the cytosol. The total cellular amount of G11alpha subunits was decreased to 21% and 59% for human and murine isoforms, respectively, and beta subunits were decreased to 68% after sustained treatment with TRH compared with controls (100%). Such data are consistent with the notion that the agonist-occupied long isoform of the rat TRH receptor may be able to partially differentiate between the endogenous (human) and exogenous (murine) G11alpha. This was not a reflection that the murine G protein was expressed but incorrectly folded as both species variants of G11alpha were solubilized equally from E2M11 membranes by sodium cholate. Using this system, we demonstrate both agonist-induced subcellular redistribution and down-regulation of G11alpha and beta subunit proteins in response to activation of a phospholipase C coupled receptor.
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PMID:Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11alpha: analysis of agonist regulation of coexpressed G11alpha species variants. 860 92


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