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 lipid composition of bovine thyroid plasma membranes was modified using the nonspecific lipid transfer protein from bovine liver. Incubation of plasma membranes with transfer protein and phosphatidylinositol-containing liposomes caused a strong, concentration dependent, inhibition of TSH-stimulated adenylate cyclase activity. Other phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid were two to four times less effective as inhibitors of TSH-stimulation. The phosphatidylinositol-induced inhibition was not reversed when more than 80% of phosphatidylinositol incorporated was removed using phosphatidylinositol-specific phospholipase C. Incorporation of phosphatidylinositol in plasma membranes provoked no significant change in the fluorescence anisotropies of the fluorophores 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(14-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), indicating that the inhibition was not due to changes in membrane fluidity. At phosphatidylinositol concentrations causing a 66% reduction in TSH-stimulated adenylate cyclase activity cholera toxin- and forskolin-stimulated activity as well as basal activity were decreased by maximally 10%. Since TSH binding to bovine thyroid plasma membranes was not affected it is suggested that phosphatidylinositol can act as a negative modulator of the TSH activation of adenylate cyclase and this probably by interfering with the coupling between the occupied TSH receptor and the stimulatory GTP-binding regulatory protein of the adenylate cyclase complex.
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PMID:Modification of TSH-stimulated adenylate cyclase activity of bovine thyroid by manipulation of membrane phospholipid composition with a nonspecific lipid transfer protein. 333 6

The TSH receptor from human thyroid plasma membranes has been solubilized in 10 mM Tris/HCl, 50 mM NaCl, pH 7.4 containing 0.5% triton X-100. Binding of [125I]TSH to the soluble receptor showed rapid and reversible kinetics and reached a maximum within 30 min at 37 degrees C, by 1 h at 25 degrees C and by 24 h 4 degrees C. Optimal pH was 7.4. The soluble receptor retained specificity with cross-reactivity only to crude hCG (0.03%). Scatchard plots were curvilinear indicating the presence of at least two binding sites. The high affinity site showed an affinity content of 1.1 X 10(9) M-1 with binding capacity of 1.3 X 10(-10) M/mg protein. TSH-binding inhibitor immunoglobulins from patients with Graves' disease inhibited [125I]TSH binding to the soluble receptor in a dose-dependent manner. NaCl inhibited the TSh binding and this was ascribed to the decrease in the receptor capacity. Trypsin, neuraminidase and phospholipase C treatment of the solubilized receptor had no effect on TSH binding. The apparent molecular weight of the receptor, determined by gel filtration of Sepharose 6B, was approximately 300 000.
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PMID:Characterization of triton-solubilized TSH receptors from human thyroid plasma membranes. 626 43

Evaluation of TSH binding to plasma membranes of porcine thyroid revealed unique sensitivity to pH and temperature. Analysis of apparent equilibrium binding yielded a linear Scatchard plot at the optimal pH of 6.0, indicating one class of binding sites. At physiological pH 7.4 a curvilinear Scatchard plot was obtained, resolved by computer analysis into two classes of binding sites of different affinities and capacities. Treatment of membranes with phospholipase C resulted in a 20% decrease in the number of high affinity sites, but no change occurred in binding affinity. In contrast, low affinity sites were not altered. To evaluate the significance of the curvilinear Scatchard plot, the kinetics of association were examined. The intrinsic Kd (kd/ka) was 0.20 nM, a value essentially equivalent to that of the high affinity binding component. The 'negative cooperativity' model of hormone binding was evaluated by examining the effect of excess unlabeled TSH on dissociation rate. Dissociation of bound 125I-labeled TSH was biphasic, and was enhanced by unlabeled hormone, regardless of whether the membranes were prelabeled at pH 6.0 or 7.4. This effect was not correlated with curvilinear Scatchard plots, and therefore not proof of negative cooperativity. Binding sites for TSH were further distinguished by their sensitivity to temperature. A van't Hoff plot of temperature dependence of the apparent Kd of the high affinity site was linear from 4 to 37 degrees C. In contrast, the apparent Kd of low affinity binding did not vary with respect to temperature. These results demonstrate that there are at least two independent binding sites for TSH on porcine thyroid plasma membranes, distinguishable by their equilibrium binding properties.
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PMID:Thyrotropin binding to porcine thyroid plasma membranes: kinetic and thermodynamic analyses. 715 94

COS-7 cells were transiently transfected with human thyrotropin receptor (TSHR) and dog A1 adenosine receptor (A1R) cDNA. TSH stimulated both inositol phosphate production and cyclic AMP (cAMP) accumulation in the cells. An A1 agonist, N6-(L-2-phenylisopropyl)adenosine (PIA), which is ineffective alone, significantly enhanced TSH-induced inositol phosphate production, but insignificantly inhibited TSH-induced cAMP accumulation was revealed by short-term treatment with the protein kinase C inhibitors, staurosporine and K252a, or long-term treatment with 12-myristate 13-acetate, suggesting that endogenous protein kinase C inhibits the A1R-mediated inhibition of the TSHR-adenylate cyclase system. In staurosporine-treated cells, the stimulatory and inhibitory permissive actions of PIA on TSH-induced phospholipase C and adenylate cyclase activation respectively were completely reversed by pretreatment with pertussis toxin whereas intrinsic TSH-induced effects were hardly affected by the toxin. The cross-talk between the signalling pathway for TSHR and that for A1R was not detected in a mixture of cells expressing either TSHR or A1R. We conclude that a single species of A1R, via pertussis-toxin-sensitive GTP-binding proteins, not only inhibits adenylate cyclase but also stimulates phospholipase C in collaboration with an activated TSHR within a single cell expressing both types of receptor.
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PMID:Intracellular cross-talk between thyrotropin receptor and A1 adenosine receptor in regulation of phospholipase C and adenylate cyclase in COS-7 cells transfected with their receptor genes. 770 64

To study the growth control of human thyroid cells in different stages of differentiation, we established two human thyroid cell lines of adenomatous goiter and papillary carcinoma. A 59-year-old female patient with adenomatous goiter was operated in September 1991, and a 27-year-old female patient with papillary carcinoma in May 1990. The thyroid cell lines were established by successive passage without cellular or genetic manipulations such as fusing other cell lines or oncogenic viral infection. These cell lines, human adenomatous goiter cells (hAG) and human papillary thyroid carcinoma cells (hPTC), exhibited a flattened polygonal shape and proliferated as a monolayer in cell culture. The doubling time of the hAG cells was 60 h in Ham's F12 medium supplemented with 10% fetal bovine serum, and that of the hPTC cells, 18 h in the same medium. Both cell lines expressed mRNA for TSH receptor and secreted cAMP into the medium during incubation with thyrotropin (TSH) at concentrations as low as 0.01 mU/mL. The effects of activators of protein kinase A (PKA), protein kinase C (PKC), tyrosine kinase (TK), and estradiol (E2) on proliferation of the hAG cells and the hPTC cells were assessed by measuring cellular DNA content in 24-well plates with diaminobenzoic acid. TSH stimulated proliferation of the hAG cells, but it inhibited proliferation of the hPTC cells. Since TSH activates two signaling pathways, the adenyl cyclase-PKA system and phospholipase C-PKC system, we tested effects of dibutylyl cAMP (dBC) and phorbol myristate 13-acetate (PMA), separately. dBC stimulated proliferation of the hAG cells, but it inhibited that of the hPTC cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Different growth control of the two human thyroid cell lines of adenomatous goiter and papillary carcinoma. 778 32

Genistein, a potent inhibitor for protein tyrosine kinase, remarkably inhibited the stimulatory action of N6-(L-2-phenylisopropyl)adenosine (PIA), an A1-adenosine receptor agonist, on thyrotropin (TSH)-induced phospholipase C activation in FRTL-5 thyroid cells. This drug also suppressed both the A1-receptor-mediated inhibition of cAMP accumulation in the cells and binding of [3H]8-cyclopentyl-1,3-dipropylxanthine, a specific antagonist for A1-receptor, to the cell membranes in a competitive manner. Adenosine-induced cAMP accumulation through A2-receptor in pertussis toxin-treated cells was also competitively antagonized by genistein. We conclude that genistein is also a competitive antagonist for P1-purinergic receptors.
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PMID:Genistein, an inhibitor of protein tyrosine kinase, is also a competitive antagonist for P1-purinergic (adenosine) receptor in FRTL-5 thyroid cells. 794 96

To characterize the role of phospholipase C (PLC)-mediated intrathyroid signal transduction by thyrotropin, we studied the effect of U-73122, an aminosteroid inhibitor of PLC-dependent activity, on TSH-activated PLC-Ca2+ and arachidonic acid (AA) signalling systems in cultured FRTL-5 rat thyroid cells. In the presence of extracellular Ca2+, TSH (0.1 microM) increased intracellular free calcium concentration ([Ca2+]i) by 63 +/- 6% with a sustained plateau phase, and AA release by 160 +/- 16%. By deletion of extracellular Ca2+, TSH induced a similar maximal [Ca2+]i increase, but the plateau phase and AA release were entirely suppressed. U-73122 (5 microM) inhibited TSH stimulation of 3H-labelled inositol trisphosphates (IP3) production by 73 +/- 3% (P < 0.01) in one study, and completely in another. U-73122 concentration-dependently blocked the TSH-induced Ca2+ increase in either the presence or absence of external Ca2+. U-73122 also showed a similar concentration-response inhibition of TSH-induced AA release. These results provide direct evidence of PLC mediation of TSH-stimulated signal transduction in FRTL-5 thyroid cells. TSH-induced external Ca2+ entry, as well as intracellular Ca2+ mobilization, is probably a PLC-mediated process. From an IP3-sensitive intracellular pool, TSH induces intracellular Ca2+ mobilization. External Ca2+ entry seems to be a prerequisite for TSH-induced AA release. U-73122 inhibition of both cytosolic Ca2+ increase and AA release further confirms [Ca2+]i dependence for TSH stimulation of AA release.
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PMID:A phospholipase C inhibitor, U-73122, blocks TSH-induced inositol trisphosphate production, Ca2+ increase and arachidonic acid release in FRTL-5 thyroid cells. 806 Oct 42

Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.
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PMID:The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families. 855 86

Thyroid cell growth and function are regulated by several hormones and growth factors that bind to cell surface receptors coupled via G proteins, Gs and Gq, to stimulation of adenylyl cyclase and phospholipase C (PLC), respectively. We created a permanently transfected FRTL-5 cell line (TG8) in which the thyroglobulin gene promoter directs expression of the cholera toxin (CT) A1 subunit (CTA1). CTA1 catalyzes ADP ribosylation of Gs alpha, which results in persistent activation of Gs alpha. Activated Gs alpha causes constitutive stimulation of adenylyl cyclase and increases levels of intracellular cAMP. Because G protein-coupled signaling pathways exhibit cross-talk, we compared TG8 cells to FRTL-5 cells transfected with the neomycin resistance gene (TG4) to determine whether constitutive stimulation of adenylyl cyclase influences the PLC pathway. PLC activity was assessed by measuring levels of total inositol phosphates (IPs) in TG4 and TG8 cells that had been preincubated with myo-[3H]inositol for 2 days. Baseline values of [3H]IP production were similar for the two cell lines. Incubation of TG4 control cells with 10(-8) M TSH, 300 microM ATP, and 100 microM norepinephrine for 60 min stimulated 2.5-, 8.1-, and 3.4-fold increases, respectively, in [3H]IP production over the control value. By contrast, there was no [3H]IP response to any of these ligands in TG8 cells. TG8 cells exhibit a decrease in [35S]adenosine 5'-(gamma-thio)triphosphate binding to their cell surface compared to TG4 control cells counterparts, but no decrease in [125I]TSH binding. Treatment of TG4 cells with 100 ng/ml CT, 50 microM forskolin, or 1 mM 8-bromo-cAMP for 2 days reproduced the loss of ligand-stimulated [3H]IP synthesis present in TG8 cells. Although levels of immunoreactive Gq alpha and Gq alpha 11 were normal in TG8 cells, sodium fluoride-induced [3H]IP production was also inhibited. Levels of immunoreactive PLC beta 3, the dominant subtype of PLC beta in FRTL-5 cells, were not altered in TG8 cells or by CT treatment of TG4 cells. These data indicate that elevated levels of cAMP can inhibit the activity of G protein-coupled PLC. Further study of this model will elucidate our understanding of the exact mechanism responsible for this interaction.
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PMID:Increased cyclic adenosine 3',5'-monophosphate inhibits G protein-coupled activation of phospholipase C in rat FRTL-5 thyroid cells. 875 35

Gonadotropin and TSH receptors represent a subgroup of seven transmembrane-spanning, G protein-coupled receptors with a large extracellular ligand-binding region. After ligand binding to their receptors, the majority of actions of gonadotropins and TSH are believed to be mediated by the cAMP-protein kinase A pathway. Although formation of inositol phosphates (IP) has been reported after stimulation of rodent gonadotropin receptors, activation of phospholipase C after ligand binding of human LH or FSH receptors has not been investigated. Human gonadotropin receptors were transiently expressed in 293 cells, and the agonist-induced stimulation of IP formation was measured. The LH receptor responded to a saturating dose of human CG (hCG) with a 5.2-fold increase of IPs whereas the FSH receptor responded to a saturating dose of FSH with only a 50% increase. On the basis of these differences and in view of the homologous nature of the two gonadotropin receptors, chimeric receptors were constructed using domain transfer to identify the regions in the human LH receptor important for phosphatidylinositol hydrolysis. Chimeric receptors containing the entire extracellular region of the FSH receptor and the seven transmembrane region plus the cytoplasmic tail of the LH receptor responded to FSH treatment with a 4.7-fold increase in IP accumulation. In contrast, the chimeric receptor with the extracellular region of the LH receptor and the TM region plus the cytoplasmic tail of the FSH receptor responded minimally (50%) to hCG treatment. When the C-terminal third (from TM V to the cytoplasmic tail) of the FSH receptor was replaced with the LH receptor sequence, the chimeric receptor still responded to FSH treatment with a large (6.2-fold) increase in IP release, similar to that of the wild type LH receptor (to hCG), suggesting that C-terminal third of the human LH receptor confers IP signaling ability. This functional domain was further divided into two areas, namely TM V to TM VI and TM VII to the cytoplasmic tail. The chimeric receptors F(I-IV)L(V-VI)F(VII-C)R and F(I-VI)L-VII-C)R, in which these two regions of the FSH receptor were replaced by the corresponding sequences of the LH receptor, responded to FSH treatment with partial increases in phosphatidylinositol hydrolysis (2.0- and 3.7-fold, respectively). Furthermore, when TM VII and the cytoplasmic tail of the LH receptor were replaced with the corresponding sequence of the FSH receptor, this chimeric receptor showed a diminished (2.0-fold) response to hCG in IP release. For all the chimeric receptor constructs analyzed, overall expression, equilibrium binding constants, and adenyl cyclase activation were not altered. Thus, unlike studies using chimeric muscarinic and dopaminergic receptors in which the second and third intracellular loops were found to be important for IP signaling, the entire C-terminal third of the human LH receptor is important for IP release. Future analysis using the chimeric receptor approach should provide new information on the structure-function relationship of gonadotropin, TSH, and other seven transmembrane-spanning receptors.
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PMID:The C-terminal third of the human luteinizing hormone (LH) receptor is important for inositol phosphate release: analysis using chimeric human LH/follicle-stimulating hormone receptors. 888 47


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