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 signal transduction pathways of the recently cloned porcine kidney calcitonin (CT) receptor were evaluated. This receptor, when stably transfected into MC-3T3 cells, avidly bound salmon CT (SCT) [dissociation constant (Kd) = 4 nM]. Incubation with SCT resulted in a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) [50% effective concentration (EC50) = 0.02 nM] in transfected cells (referred to as PC-1 cells). Binding kinetics and cAMP dose response relationships were similar to those of the native receptor in LLC-PK1 cells. PC-1 cells also responded to calcitonin gene-related peptide (CGRP), but the EC50 value for cAMP accumulation was more than three orders of magnitude higher than for SCT. Exposure of PC-1 cells to SCT (5 nM to 1 microM) produced a dose-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]i), whereas CGRP did not. The initial rise in [Ca2+]i was not dependent on extracellular Ca2+, suggesting that SCT induced release of Ca2+ from intracellular stores. SCT also increased inositol trisphosphate production in PC-1 cells. In conclusion, the cloned, transfected porcine CT receptor functionally couples to and activates both adenylyl cyclase and phospholipase C. This dual coupling is also a characteristic of the parathyroid hormone receptor, which has significant homology in amino acid sequence with the CT receptor.
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PMID:A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C. 132 Mar 32

We use a sensitive biotin polarity assay to survey the surface distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins in five model epithelial cell lines derived from different species (dog, pig, man) and tissues, i.e., kidney (MDCK I, MDCK II, LLC-PK1) and intestine (Caco-2 and SK-CO15). After biotinylation of apical or basolateral surfaces of confluent monolayers grown on polycarbonate filters, GPI-anchored proteins are identified by their shift from a Triton X-114 detergent-rich phase to a detergent-poor phase in the presence of phosphatidylinositol-specific phospholipase C. All GPI-anchored proteins detected (3-9 per cell type, at least 13 different proteins) are found to be apically polarized; no GPI-anchored protein is observed preferentially localized to the basal surface. One of the GPI-anchored proteins is identified as carcinoembryonic antigen (CEA). Survey of MDCK II-RCAr, a mutant cell line with a pleiotropic defect in galactosylation of glycoproteins and glycolipids (that presumably affects GPI anchors) also reveals an apical polarization of all GPI-anchored proteins. In contrast, analysis of MDCK II-ConAr (a mutant cell line with an unknown defect in glycosylation) revealed five GPI-anchored proteins, two of which appeared relatively unpolarized. Our results indicate that the polarized apical distribution of GPI-anchored proteins is highly conserved across species and tissue-type and may depend on glycosylation.
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PMID:Preferred apical distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype. 213 77

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.
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PMID:Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor. 214 82

LLC-PK1 cells have been shown to possess vasopressin (VP) receptors (V2 type) that are coupled to adenyl cyclase to generate adenosine 3,5'-cyclic monophosphate (cAMP). To determine whether VP also stimulates phosphoinositide (PI) hydrolysis to generate inositol phosphate (IP) and diacylglycerol (DAG) messenger system in LLC-PK1 cells, we measured the release of IP in LLC-PK1 cells in the absence and presence of various concentrations of VP. In addition, we also determined the effect of an increase in osmolality of the incubation medium on VP-stimulated PI hydrolysis in LLC-PK1 cells. The methods involved the incubation of LLC-PK1 cells with [3H]inositol for its incorporation into membrane PI and the measurement of the release of [3H]IP in the presence of LiCl which prevents dephosphorylation. The osmolality of the incubation media was increased from 300 to 600, 900, and 1,200 mosmol/kgH2O by the addition of NaCl and urea. In an isosmotic incubation medium, VP (10(-8) M) produced a 100% increase in PI hydrolysis in LLC-PK1 cells. The effect was much greater at higher concentrations of the hormone. There was no effect of osmolality in VP-stimulated PI hydrolysis in LLC-PK1 cells up to 600 mosmol/kgH2O, but PI hydrolysis decreased significantly when the osmolality of the incubation medium was increased to 900 or 1,200 mosmol/kgH2O. Our results suggest that in LLC-PK1 cells, VP stimulates PI hydrolysis probably through VP receptors that are coupled to phospholipase C. Furthermore, VP-stimulated PI messenger system in LLC-PK1 cells is influenced by osmolality of the extracellular fluid.
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PMID:Vasopressin stimulates phosphoinositide hydrolysis in LLC-PK1 cells. 284 98

Membrane proteins can be attached to the plasma membrane in several ways. Recently, a mechanism has been described, by which a number of cell surface proteins are anchored to the exoplasmic side of the plasma membrane by covalent linkage to glycosyl-phosphatidylinositol (GPI). The growth properties of renal epithelial cells in tissue culture enable free access to apical cell surface and brush border membrane proteins. To study the nature of membrane anchoring of apical plasma membrane enzymes in cultured renal epithelial cells, confluent LLC-PK1, OK, NRK, and MDCK epithelia were treated in tissue culture dishes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the PI-PLC-specific release into the tissue culture medium of the apical membrane enzymes alkaline phosphatase (AP), gamma-glutamyl transpeptidase, leucine aminopeptidase, trehalase, and maltase was determined. Of the five enzymes tested, AP and trehalase, already described as GPI-anchored membrane proteins, were specifically released by PI-PLC from intact cell monolayers. Of the four cell lines investigated, LLC-PK1 cells express AP and trehalase which were released by PI-PLC. In OK cells, which lack AP activity, only trehalase was found to have PI-PLC-releaseable enzyme activity. MDCK cells, on the other hand, express AP activity, releaseable by PI-PLC, but no trehalase activity. In studies on the time course of synthesis and reinsertion of AP into the apical membrane of LLC-PK1 cells after removal by PI-PLC, a 60% recovery of AP activity was obtained only after 7 days. Analysis of protein release by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of culture supernatants after surface labeling with biotin and subsequent Western blotting with streptavidin revealed four protein bands at approximately 130, 90, 30, and 20 kD in LLC-PK1 cells and five GPI-anchored proteins at 110, 85, 65, 40, and 26 kD in OK cultures. The finding of a PI-PLC-specific release of apical membrane enzymes from renal tubular cell lines of different species (pig, opossum, rat, and dog) and of different nephron origin indicates a high conservation of the GPI anchor of renal brush border membrane proteins and further proves the high degree of differentiation retained by the cell lines in tissue culture. In addition, this method may provide a possible tool for isolating GPI-anchored apical membrane proteins from intact epithelial monolayer cultures.
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PMID:Selective release of apical membrane enzymes from cultured renal epithelia by phosphatidylinositol-specific phospholipase C. 750 39

We showed previously that a single species of cloned PTH/PTH-related peptide (PTHrP) receptors, when stably expressed in LLC-PK1 kidney cells, couples to multiple second messenger signals and biological responses. To address the linkages of individual messenger signals to specific biological responses in these cells, we examined the relations among PTH/PTHrP receptor expression, PTH-activated phospholipase C (PLC) and adenylyl cyclase, and PTH-regulated phosphate transport in LLC-PK1 cells that stably express cloned rat PTH/PTHrP receptors. Among 18 such subclones, PTH stimulation of intracellular cAMP accumulation was nearly equivalent, despite differences in receptor density ranging from 20,000-400,000 sites/cell. In contrast, activation of PLC by PTH was directly and continuously dependent upon receptor density. PTH-stimulated phosphate uptake also was strongly dependent upon receptor expression, correlated well with PLC activity, was mimicked by active phorbol esters but not by cAMP analogs or forskolin, and was strikingly inhibited by the protein kinase C inhibitor, staurosporine. The peptide analog [Arg2]human PTH-(1-34), which significantly stimulated cAMP accumulation but failed to activate PLC, also did not increase phosphate uptake. We conclude that in LLC-PK1 cells, PTH-modulated PLC activation, unlike adenylyl cyclase activation, is strongly dependent upon PTH/PTHrP receptor density. This feature is reflected in the analogous relation between receptor density and PTH regulation of phosphate uptake, which appears to be mediated via a PKC-dependent pathway in these transfected cells. The results suggest that regulation of PTH/PTHrP receptor expression on target cells may provide a mechanism for altering the character as well as the magnitude of the signaling response to the hormone.
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PMID:Parathyroid hormone (PTH)/PTH-related peptide receptor density modulates activation of phospholipase C and phosphate transport by PTH in LLC-PK1 cells. 764 96

Angiotensin II (ANG II) receptors of the AT1 subtype are present on the apical and basolateral membranes of renal proximal tubule cells. Cells of the proximal tubulelike cell line, LLC-PK1/Cl4, were transfected with an expression plasmid containing cDNA encoding the rabbit AT1 ANG II receptor. In transfected cells, specific binding of 125I-ANG II was detected on both apical and basolateral membranes; wild-type LLC-PK1/Cl4 cells did not express ANG II receptors. In transfected cells, apical or basolateral ANG II increased both S6 kinase activity and incorporation of [3H]leucine. In cells pretreated with pertussis toxin, the stimulatory effect of apical or basolateral ANG II on [3H]leucine incorporation was abolished. In contrast, ANG II did not affect mitogenesis, determined by [3H]thymidine incorporation. Apical or basolateral ANG II (10(-6) M) stimulated phosphoinositide turnover by 13.4 +/- 4.4% (n = 8) and 16.3 +/- 4.2% (n = 9), respectively. The activity of protein kinase C, determined by phosphorylation of a specific protein kinase C peptide substrate, was also stimulated by ANG II in transfected cells. Apical or basolateral ANG II had no significant effect on cellular adenosine 3',5'-cyclic monophosphate levels. In permeabilized transfected cells, apical ANG II (10(-6) M) inhibited the phosphorylation of a specific peptide substrate of protein kinase A; lower apical concentrations or basolateral ANG II were without significant effect. These results indicate that AT1 ANG II receptors sort to both apical and basolateral membranes in renal epithelial cells and are coupled to activation of phospholipase C. ANG II stimulates protein synthesis by binding to either apical or basolateral receptors; this effect requires coupling to G proteins and may be mediated by activation of S6 kinase. Because high concentrations of ANG II exist in proximal tubule, binding to apical and basolateral receptors may regulate proximal tubule cell growth under physiological conditions.
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PMID:Signaling and growth responses of LLC-PK1/Cl4 cells transfected with the rabbit AT1 ANG II receptor. 773 40

The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured renal LLC-PK1 epithelial cells were estimated by confocal laser microscopy. No difference between the resting mean [Ca2+]n and [Ca2+]c was found. During stimulation with maximal effective concentrations of arginine vasopressin (AVP) or the purinergic agonist ATP, the transient Ca2+ rise was followed mostly by a decline to basal levels. A differential rise could be observed when the increase in [Ca2+]n attained higher values than [Ca2+]c. In 50-60% of the cells, epidermal growth factor (EGF) also induced a transient Ca2+ rise, and a differential increase ([Ca2+]n > [Ca2+]c) was found. The G protein-linked stimuli AVP and ATP were however quantitatively much more efficacious at stimulating the [Ca2+]n and [Ca2+]c increases than was EGF. To investigate whether AVP, ATP, and EGF released Ca2+ from distinct or overlapping stores, the agonists were sequentially added. AVP and ATP applied after EGF in Ca(2+)-free medium elicited an increase in [Ca2+]n and [Ca2+]c that was not significantly lower than the release of Ca2+ in control cells without EGF prestimulation. Similarly, the amplitude of the Ca2+ responses attained by EGF in cells prestimulated by ATP or AVP was comparable to the response in naive cells. Neither EGF, ATP, nor AVP evoked a Ca2+ signal after thapsigargin treatment, indicating that the intracellular Ca2+ pools stimulated by all these agonists are part of the thapsigargin-sensitive Ca2+ pools. In contrast, when ATP was applied after AVP in Ca(2+)-containing as well as in Ca(2+)-free solutions, the Ca2+ transients were lower as compared with the response without preincubation. No differential rise could be found in Ca(2+)-free conditions. An explanation could be the use of different phospholipase C isozymes by the different receptor types, which possibly gives rise to the mobilization of different Ca2+ pools.
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PMID:Intracellular Ca2+ signaling induced by vasopressin, ATP, and epidermal growth factor in epithelial LLC-PK1 cells. 823 22

We investigated the effect of extracellular ATP on the interaction of epidermal growth factor (EGF) with its receptor in cultured renal epithelial cells, LLC-PK1. Pretreatment with ATP, but not adenosine, inhibited the binding of 125I-labeled EGF. The inhibition demonstrated by ATP resulted from a decrease in the affinity of EGF receptors for its ligand, with no change in the number of EGF receptors. Incubation of phorbol 12-myristate 13-acetate (PMA) for 30 min mimicked the ATP-mediated inhibition. On the other hand, prolonged pretreatment with PMA, which leads to disappearance of protein kinase C activity, reversed the inhibition. In addition, pretreatment with the protein kinase C inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine prevented the ATP-mediated inhibition. ATP triggered an increase in inositol 1,4,5-trisphosphate levels and translocation of protein kinase C from cytosol to membranes, consist with the stimulation of phospholipase C and the activation of protein kinase C. These results demonstrate that extracellular ATP attenuates the ligand binding affinity of EGF receptor via the stimulation of phospholipase C, leading to the activation of protein kinase C in the LLC-PK1 cells.
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PMID:Extracellular ATP-induced regulation of epidermal growth factor signaling in cultured renal LLC-PK1 cells. 847 24

The metabotropic glutamate receptors (mGluRs) share no sequence homology and show different structural features compared with most other G protein-coupled receptors (GPCRs). In particular, some isoforms of the phospholipase C (PLC)-coupled mGluRs (mGluR1a, mGluR5a, and mGluR5b) have a surprisingly long carboxyl-terminal intracellular domain of more than 350 residues, whereas the splice variants mGluR1b and mGluR1c have a much shorter carboxyl terminus. In the current study, the different splice variants of mGluR1 were expressed in porcine kidney epithelial (LLC-PK1) or the human embryonic kidney (HEK 293) cells, and their levels of expression were examined with the use of Western blot analysis. Expression of the short isoforms mGluR1b and mGluR1c did not modify the basal inositol phosphate production. In contrast, expression to similar levels of mGluR1a resulted in a 2-fold increase in the basal inositol phosphate formation. This increase in basal PLC activity was due to neither the presence of a low concentration of glutamate in the incubation medium nor a modification of the PLC pathway, resulting, for example, from the constant activation of mGluR1a++ by glutamate during the culture. Surprisingly none of the known competitive antagonists of mGluR1 inhibited the basal PLC activity, indicating that none of these molecules act as inverse agonists. Taken together, these results indicate that the long carboxyl-terminal domain confers a small agonist-independent activity to mGluR1. This indicates that, as already observed for other GPCRs, little constitutive activity of wild-type mGluRs can be detected. Our results also add to the splice variants and further suggest that the long carboxyl-terminal domain of mGluR1a confers better coupling efficiency to the G proteins.
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PMID:Changes in the carboxyl-terminal domain of metabotropic glutamate receptor 1 by alternative splicing generate receptors with differing agonist-independent activity. 864 81


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