UNIPROT:P00750 - FACTA Search

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Query: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
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PMID:Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. 131 32

Five rat thyroid cell lines were tested for the expression of the cell surface receptor for urokinase type plasminogen activator (uPA). All tested lines were found to bind uPA, but transformed 1-5G and Ki-Mol cells, which are also high uPA producers, bound at least ten times more uPA, as compared to non-producers, 'normal' TL5 cells. Moreover, it was possible to remove membrane-bound uPA by treating the cells with phosphatidylinositol-specific phospholipase C, suggesting that rat uPAR, like its human counterpart, is linked to the membrane by a glucosyl-phosphatidylinositol anchor. The specificity of the binding was tested by competition with three different synthetic peptides corresponding to amino acids 14-37 of human, rat and mouse uPA. The results indicate also that the receptor binding region of rat uPA is located within the growth factor domain of the molecule and that its expression may be dependent on the transformed state of the cells.
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PMID:The receptor for the plasminogen activator of urokinase type is up-regulated in transformed rat thyroid cells. 132 34

The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.
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PMID:Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens. 132 97

Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells and stimulates haematopoiesis in vitro. We report here that primary human bone marrow cultures contain bFGF and express heparin-like bFGF binding sites on the cell surface and in the extracellular matrix (ECM). bFGF bound predominantly to a 200-kD cell surface heparan sulfate proteoglycan (HSPG), which was also found in conditioned medium. bFGF was released from bone marrow cultures by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) and, less efficiently, by plasmin. Solubilized bFGF was found as a complex with the 200-kD HSPG. The complex was biologically active as shown by its ability to stimulate plasminogen activator production in bovine aortic endothelial cells. bFGF-HSPG complexes of bovine endothelial cells, however, were not released by PI-PLC. While only trace amounts of the bFGF-binding 200-kD HSPG were released spontaneously from bone marrow cultures, incubation with PI-PLC solubilized almost all of the 200-kD HSPG. The HSPG could be metabolically labeled with ethanolamine or palmitate, which was partially removed by treatment with PI-PLC. These findings indicate linkage of the HSPG to the cell surface via a phosphatidylinositol anchor. Plasmin released the 200-kD HSPG less efficiently than PI-PLC. We conclude that HSPGs of human bone marrow serve as a reservoir for bFGF, from which it can be released in a biologically active form via a dual mechanism; one involving a putative endogenous phospholipase, the other involving the proteolytic cascade of plasminogen activation.
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PMID:Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan. 165 37

The importance of cell-associated plasminogen activation in the extracellular matrix degradation processes is becoming increasingly evident. To elucidate the modulators of net plasminogen activation on the cell surface, we have recently established an assay system. Using this system, we examined the effects of several candidate modulators on cell surface plasminogen activator in the human fibrosarcoma cell line HT-1080 and the SV40-transformed human lung fibroblast cell line WI-38 VA 13 2RA. Although the majority of the candidates had no effect or a selective effect on either cell line, only retinoic acid markedly enhanced cell surface plasminogen activator activity in both HT-1080 and WI-38 VA13 2RA cells in a time-dependent manner. The effect of retinoic acid was neutralized by actinomycin D. The enhanced activity was inhibited by anti-uPA IgG and by pretreatment with phosphatidylinositol-specific phospholipase C. These findings suggest that retinoic acid increases the amount of receptor-bound uPA via de novo synthesis, and that it plays an important role in modulating cell-associated plasminogen activation.
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PMID:Retinoic acid enhances plasminogen activation on the cell surface. 857 37

Urokinase-type-plasminogen activator (uPA) and its receptor are localized in the vessel wall where they are involved in cellular activation and remodelling processes. Besides the cell surface glycolipid (GPI)-anchored urokinase receptor (uPAR), which binds uPA with high affinity, recent evidence points to the existence of soluble uPAR (suPAR), as well. In the present study, the origin, binding mechanism, and cellular effects of suPAR were examined. Under basal conditions human vascular smooth muscle cells (HVSMC), human umbilical vein endothelial cells (HUVEC), and monocytic cells released 0.1 to 2 ng/mL suPAR, which was increased twofold to fivefold after phorbol ester (PMA) stimulation, as measured by a function-dependent enzyme-linked immunosorbent assay (ELISA). suPAR alone did not bind to HVSMC or HUVEC, but reduced cellular uPA binding by 50% to 70%. However, after removal of GPI-uPAR with phosphatidylinositol-specific phospholipase C, suPAR dose-dependently increased uPA binding by fourfold to fivefold. This increase in binding was completely inhibited by vitronectin (VN) and by a monoclonal antibody against VN, but not by other matrix proteins or antibodies. Thus, VN-mediated uPA binding to cells was regulated by the ratio of soluble to surface-associated uPAR. In a uPAR-deficient cell line (LM-TK-), suPAR increased uPA binding up to 10-fold, whereas the truncated receptor lacking the amino-terminal uPA-binding domain was ineffective. The formation of a ternary uPA/suPAR/VN-complex on the cell surface and the free extracellular matrix could be inhibited by a monoclonal antibody against VN, as well as by plasminogen activator inhibitor-1 (PAI-1). Moreover, VN-mediated binding of the uPA/suPAR-complex led to a fivefold increase in plasminogen activator activity. Through this novel pathway, VN concentrates the uPA/suPAR-complex to cell surfaces and extracellular matrix sites, leading to the accumulation of plasminogen activator activity required for cell migration and tissue remodelling processes.
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PMID:Vitronectin concentrates proteolytic activity on the cell surface and extracellular matrix by trapping soluble urokinase receptor-urokinase complexes. 951 28

The plasminogen activator system has been implicated in the modulation of the response to vascular injury. Although urokinase-type plasminogen activator (uPA) and its receptor (uPAR) may enhance matrix degradation as well as migration and invasion by smooth muscle cells (SMCs), their roles in cell adhesion are uncertain. Therefore, we examined the ability of uPA and uPAR to modulate adhesion of cultured human vascular SMCs to various matrices. We demonstrated a dose-dependent stimulation of adhesion by single-chain uPA (scuPA) to vitronectin (maximum 1.55-fold [+/-0. 04-fold] increase, 10 nmol/L, P<0.002) but not to laminin, collagen I, or collagen IV. Baseline adhesion to vitronectin was completely inhibited by both EDTA and RGD peptide but was restored to >40% of control in the presence of scuPA (P=0.001 and 0.046, respectively). Adhesion to vitronectin was also significantly enhanced by the amino-terminal fragment of uPA (P=0.007) and two-chain, high-molecular-weight uPA (P<0.01) but not by the low-molecular-weight fragment of uPA, which lacks the receptor-binding domain. Aprotinin, a plasmin inhibitor, had no effect on baseline or scuPA-stimulated adhesion, suggesting a plasmin-independent process. Preincubation of scuPA with soluble uPAR inhibited scuPA stimulation of adhesion by 88+/-14% (P=0.01), as did pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes glycophosphatidylinositol-anchored proteins, including uPAR. Antibodies to both alphavbeta3 and alphavbeta5 integrin inhibited baseline adhesion but not scuPA stimulation. Finally, coating plates with scuPA alone enabled cell adhesion, which could be inhibited by both soluble uPAR and anti-uPAR antibodies. These data suggest that uPA stimulates adhesion of SMCs specifically to vitronectin and that it is mediated by an interaction with uPAR. Upregulation of both proteins after vascular injury may facilitate migration through stimulation of both matrix degradation and cell adhesion.
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PMID:Urokinase receptor-dependent upregulation of smooth muscle cell adhesion to vitronectin by urokinase. 984 76

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

The signal transduction involved in the purinergic stimuli-induced activation of protein kinase C (PKC) in CHO-K1 cells was investigated. Purinergic stimuli such as adenosine triphosphate and uridine triphosphate induced a transient translocation of PKC epsilon, gamma, and delta from the cytoplasm to the plasma membrane. These translocations were blocked by an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), but not by an inhibitor of phosphatidylcholine-specific PLC. A diacylglycerol (DAG) analogue also induced reversible translocations of PKC gamma, epsilon, and delta from the cytoplasm to the plasma membrane, while the calcium ionophore A23187 caused a similar translocation of only the gamma subtype. These results confirm that the hydrolysis of phosphatidylinositol-2-phosphate by PLC and the subsequent generation of DAG and increase in Ca(2+ )are involved in the purinergic stimuli-induced translocation of PKC. A DAG antagonist, 1-o-hexadecyl-2-o-acetyl-glycerol, blocked the DAG analogue-induced translocations of all PKC subtypes tested but failed to inhibit the purinergic stimuli-induced translocations of PKC epsilon and gamma. The DAG antagonist could not block the ATP- and UTP-induced translocation of PKC epsilon even in the absence of extracellular Ca(2+). Co-application of the DAG antagonist and a phospholipase A(2) (PLA(2)) inhibitor such as aristolochic acid, arachidonyltrifluoromethyl ketone, or bromoenol lactone inhibited the purinergic receptor-mediated translocation of PKC epsilon although each PLA(2) inhibitor alone did not block the translocation. In contrast to the epsilon subtype, ATP-induced translocation of PKC gamma was observed in the presence of both the PLA(2) inhibitor and the DAG antagonist. However, it is noteworthy that re-translocation of PKC gamma was hastened by the PLA(2) inhibitor. Furthermore products of PLA(2), such as lysophospholipids and fatty acids, induced the translocation of PKC gamma and epsilon in a dose dependent manner, but not delta. These results indicate that, in addition to PLC and DAG, PLA(2) and its products are involved in the purinergic stimuli-induced translocation of PKC epsilon and gamma in CHO-K1 cells. Each subtype of PKC in CHO-K1 cell is individually activated in response to a purinergic stimulation.
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PMID:Phospholipase A(2) and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells. 1072 17

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.
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PMID:The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C. 1154 56

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