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)

Parathyroid hormone (PTH) activates both adenylate cyclase and phospholipase C in target cells, and cloned PTH/PTH-related protein (PTHrP) receptor can mediate both responses when expressed in host cells such as LLC-PK1 renal epithelial cells. Because calcitonin (CT) is known to augment 70-kDa heat shock protein (HSP70) mRNA by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism in LLC-PK1 cells, we examined regulation of HSP70 transcription by PTH in these cells. Like CT, human PTH-(1-34) [hPTH-(1-34); 10(-10) to 10(-7) M)] increased porcine HSP70 mRNA and human HSP70 promoter-chloramphenicol acetyltransferase (CAT) expression within 4 h in LLC-PK1 cells that stably express > or = 100,000 PTH/PTHrP receptors per cell. The effect of PTH on HSP70 mRNA was not mimicked by cAMP analogues, forskolin, phorbol esters, Ca2+ ionophores, or alpha-thrombin; was insensitive to pertussis toxin; and was not due to increased mRNA stability. The upregulation of HSP70 gene transcription by hPTH (and CT) was clearly observed even after deletion of the functional heat shock consensus element in the promoter region of the human HSP70/CAT reporter. Upregulation of HSP70 transcription via endogenous PTH receptors also was observed in the osteoblastic cell lines SaOS-2 and ROS 17/2.8. Regulation of HSP70 gene transcription by PTH may be a common cellular response to the hormone, which, in some cells, may not be mediated by activation of adenylate cyclase or protein kinase C.
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PMID:Regulation of HSP70 by PTH: a model of gene regulation not mediated by changes in cAMP levels. 876 37

To define the structural requirements of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor necessary for activation of phospholipase C (PLC), receptors with random mutations in their second cytoplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKKY (amino acids 317-320) was replaced with DSEL had little or no PTH-stimulated PLC activity when expressed transiently in COS-7 cells, but it retained full capacity to bind ligand and to generate cAMP. This phenotype was confirmed in LLC-PK1 cells stably expressing the DSEL mutant receptor, where both PTH-stimulated PLC activity and sodium-dependent phosphate co-transport were essentially abolished. Individual mutations of these four residues point to a critical role for Lys-319 in receptor-G protein coupling. PTH-generated IPs were reduced to 27 +/- 13% when K319E, compared with the WT receptor, and PLC activation was fully recovered in a receptor revertant in which Glu-319 in the DSEL mutant cassette was restored to the WT residue, Lys. Moreover, the WT receptor and a mutant receptor in which K319R had indistinguishable properties, thus suggesting that a basic amino acid at this position may be important for PLC activation. All of these receptors had unimpaired capacity to bind ligand and to generate cAMP. To ensure adequacy of Galphaq-subunits for transducing the receptor signal, Galphaq was expressed in HEK293 and in LLC-PK1 cells together with either WT receptors or receptors with the DSEL mutant cassette. PTH generated no inositol phosphates (IPs) in either HEK293 or LLC-PK1 cells, when they expressed DSEL mutant receptors together with Galphaq. In contrast, PTH generated 2- and 2. 5-fold increases in IPs, respectively, when these cells co-expressed both the WT receptor and Galphaq. Thus, generation of IPs by the activated PTH/PTHrP receptor can be selectively abolished without affecting its capacity to generate cAMP, and Lys-319 in the second intracellular loop is critical for activating the PLC pathway. Moreover, alpha-subunits of the Gq family, rather than betagamma-subunits, transduce the signal from the activated receptor to PLC, and the PLC, rather than the adenylyl cyclase, pathway mediates sodium-dependent phosphate co-transport in LLC-PK1 cells.
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PMID:Mutations in the second cytoplasmic loop of the rat parathyroid hormone (PTH)/PTH-related protein receptor result in selective loss of PTH-stimulated phospholipase C activity. 905 74

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis added to a culture of LLC-PK1 cells inhibited cell growth by 40%. In contrast with normal cells, the cells cultured in the presence of PI-PLC showed needle-like appendages which seemed to have been formed due to portions of the cell remaining adhered to the culture dish as the cell shrank. When LLC-PK1 cells were treated with PI-PLC, significant amounts of alkaline phosphatase and alkaline phosphodiesterase I were released specifically from the apical surface of the LLC-PK1 cells. Furthermore, PI-PLC treatment caused a delay of enzyme production and dome formation. These data indicate that glycosyl-phosphatidylinositol (GPI)-anchored proteins on the surface of LLC-PK1 cells are important in cell growth and differentiation. Also, the combined use of LLC-PK1 cells and PI-PLC of B. thuringiensis is effective for investigating the function of GPI-anchor proteins.
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PMID:Growth inhibition, morphological change, and ectoenzyme release of LLC-PK1 cells by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis. 917 52

Parathyroid hormone (PTH) activates multiple intracellular effectors, including adenylyl cyclase (AC) and phospholipase C (PLC), via a single receptor [PTH/parathyroid hormone-related protein receptor (PTHR)] expressed in bone and kidney. Homologous desensitization of PTHR signaling occurs, but the relative importance of reduced receptor expression vs. impaired receptor-effector coupling in this process remains unclear. It also is not known if AC and PLC responses to PTH are desensitized independently or interdependently. In LLC-PK1 cells that expressed transfected wild-type PTHRs, PTH caused dose- and time-dependent desensitization of both the AC and PLC-responses to PTH without altering PTHR expression. Desensitization of AC was blocked in mutant cells resistant to adenosine 3',5'-cyclic monophosphate but not when cells expressed mutant PTHRs with defective PLC coupling. Desensitization of PLC was unaffected by PKA blockade, partially mimicked by phorbol ester, and not reproduced by agents that selectively activated AC. The finding that homologous PTHR desensitization in LLC-PK1 cells is signal specific suggests that prior exposure of other cells to PTH also may induce discordant regulation of subsequent PTHR signaling, altering the character as well as the intensity of the hormonal response.
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PMID:Mechanisms of homologous and heterologous desensitization of PTH/PTHrP receptor signaling in LLC-PK1 cells. 927 93

LLC-PK1, an epithelial cell line derived from the kidney proximal tubule, was used to study the ability of the G protein alpha-subunit, G alpha q, to regulate cell differentiation. A constitutively active mutant protein, alpha qQ209L, was expressed using the LacSwitch-inducible mammalian expression system. Induction of alpha qQ209L expression with isopropyl-beta-D-thiogalactopyranoside (IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold. Increasing concentrations of IPTG progressively inhibited the activity of two differentiation markers, Na(+)-dependent hexose transport and alkaline phosphatase activity. Induction of alpha qQ209L expression also caused a change from an epithelial to a spindle-shaped morphology. The effects of alpha qQ209L expression on cell differentiation were similar to those observed with 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. However, protein kinase C (PKC) levels were downregulated in TPA-treated cells but not in alpha qQ209L-expressing cells, suggesting that the regulation of PKC by G alpha q may be different from regulation by TPA. Interestingly, the PKC inhibitor GF-109203X did not inhibit the effect of IPTG on the development of Na(+)-dependent hexose transport in alpha qQ209L-expressing cells. These data implicate PKC delta and PKC epsilon in the pathway used by G alpha q to block the development of Na(+)-dependent hexose transport in IPTG-treated cells.
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PMID:Inhibition of cell differentiation by G alpha q in the renal epithelial cell line LLC-PK1. 957

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.
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PMID:Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor. 964 99

The carboxyl(C)-truncated human (h) PTH (hPTH) analog hPTH(1-31), which activates adenylyl cyclase (AC), but not protein kinase C, in rat osteosarcoma cells, exerts an anabolic effect on rat bone in vivo similar to that of hPTH(1-34). It has been proposed, therefore, that this action of PTH(1-34) is mediated exclusively by stimulation of AC via the rat type-1 PTH/PTH-related peptide (PTHrP) receptor (PTH1R). To determine whether this selective signaling pattern also might be a property of the hPTH1R, we studied signal transduction via heterologously expressed hPTH1Rs in response to activation by hPTH(1-34), hPTH(1-31), and a C-truncated analog that does not increase rat bone mass in vivo, hPTH(1-30). In porcine LLC-PK1 cells that stably expressed recombinant hPTH1Rs, these three peptides activated AC identically (EC50 = 1-2 nM). In cells with comparable expression of rat PTH1Rs, AC activation by hPTH(1-34) and hPTH(1-31) again was identical, whereas full activation by hPTH(1-30) required higher concentrations (EC50 = 10 nM vs. 1 nM). Surprisingly, hPTH(1-31) fully stimulated phospholipase C (PLC), via both species of PTH1Rs, with potency that was similar (hPTH1Rs) or slightly reduced (rat PTH1Rs), relative to that of hPTH(1-34). hPTH(1-30), however, was 5-fold less potent than hPTH(1-34) in activating PLC via hPTH1Rs and showed weak and only partial activity via the rat PTH1R. Comparable results were obtained when human and rat PTH1Rs were transiently expressed heterologously in COS-7 cells or homologously in HEK 293 and UMR 106-01 cells, respectively. Binding affinities of these C-truncated peptides to human and rat PTH1Rs were concordant with their relative potencies in activating PLC. We conclude that hPTH(1-31) and, to a lesser extent, hPTH(1-30) can activate PLC, as well as AC, via both rat and human PTH1Rs. Accordingly, a role for PLC activation in the anabolic action of PTH in vivo cannot be excluded.
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PMID:Type-1 parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors activate phospholipase C in response to carboxyl-truncated analogs of PTH(1-34). 975 12

Parathyroid hormone (PTH) activates PTH/PTH-related peptide-related receptors (PTHRs) to stimulate both adenylyl cyclase (AC) and phospholipase C (PLC). How these parallel signals mediate specific cellular and tissue responses to PTH, such as the complex anabolic versus catabolic actions of PTH on bone, remains unsettled. Previous studies of PTHR signaling and function employed mainly rodent or other cell lines that express endogenous PTHRs and, possibly, alternate species of PTH receptors. To preclude confounding effects of such receptors, we stably expressed recombinant human PTHRs (hPTHRs) at different levels of surface density in LLC-PK1 porcine renal epithelial cells that lack endogenous PTH responsiveness. hPTH(1-34) induced concentration-dependent activation of both AC and PLC via transfected hPTHRs. Maximal intensity of each signal increased with receptor density, but more hPTHRs were required for PLC than for AC activation. Coupling to AC was saturated at receptor densities too low to detect sustained PLC activation. hPTH(3-34), found by others to be a PLC/protein kinase C (PKC)-selective peptide in rat cells, did not activate PLC via human (or rat) PTHRs under conditions (1 microM peptide, 106 hPTHRs/cell) where hPTH(1-34) stimulated PLC severalfold. Other cellular responses that require PKC activation in these cells, such as sodium-dependent phosphate transport and cAMP-independent secretion of plasminogen activator, were induced by PTH(1-34) but not by hPTH(3-34) or hPTH(7-34). We conclude that amino-truncated PTH analogs reported to activate PKC cannot directly activate phosphatidylinositol-specific PLC via the human or rat PTHR and therefore that PTH receptors may access alternate, PLC-independent pathways of PKC activation in some target cells. The relative intensity of AC and PLC signaling via the hPTHR may be strongly regulated by changes in its surface expression.
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PMID:Dual signaling and ligand selectivity of the human PTH/PTHrP receptor. 989 61

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29-32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1-34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restored when a binding-enhancing substitution (Glu(19) --> Arg(19)) was placed within hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(19)]hPTH-(1-28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28-31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1-34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.
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PMID:Amino-terminal modifications of human parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type 1 PTH receptor: implications for design of signal-specific PTH ligands. 1052 Dec 52

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1 cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCalpha from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCalpha in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2 inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCalpha in OK, but not in LLC-PK1, cells. The activation of PKCalpha in OK cells by NO is associated with inhibition of Na+-K+-ATPase.
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PMID:Nitric oxide activates PKCalpha and inhibits Na+-K+-ATPase in opossum kidney cells. 1060 Sep 32


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