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:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Drug-resistant tumor cells actively extrude a variety of chemotherapeutic agents by the action of the multi-drug resistance (MDR1) gene product, the plasma membrane P-glycoprotein. In this report we show that the expression of the human MDR1 gene in cultured Sf9 insect cells via a baculovirus vector generates a high activity vanadate-sensitive membrane ATPase. This ATPase is markedly stimulated by drugs known to interact with the P-glycoprotein, such as vinblastine and verapamil, and the ability of the various drugs to stimulate the ATPase corresponds to their previously observed affinity for this transporter. The drug-stimulated ATPase is not present in uninfected or mock-infected Sf9 cells, and its appearance correlates with the appearance of the MDR1 gene product detected with a monoclonal anti-MDR protein antibody and by labeling with 8-azido-ATP. The drug-induced ATPase requires magnesium ions, does not utilize ADP or AMP as substrates, exhibits a half-maximal activation at about 0.5 mM MgATP, and its maximal activity (about 3-5 mumol/mg MDR protein/min) approaches that of the well characterized ion transport ATPases. These results provide the first direct demonstration of a high capacity drug-stimulated ATPase activity of the human multidrug resistance protein and offer a new and simple assay for the investigation of functional interactions of various drugs with this clinically important enzyme.
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PMID:Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. 134 44

Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.
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PMID:Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein. 137 98

B16 mouse melanoma cells are grown inhibited by cyclic AMP or by retinoic acid (RA). However, the combination of these two agents results in less growth inhibition than either agent alone. In order to investigate this interaction, cells were selected for resistance to 8-bromo-cyclic AMP-induced growth inhibition. Two clones (3 and 7) which demonstrated significant resistance were isolated. When these two clones were treated with retinoic acid (RA) it was observed that they also exhibited different degrees of resistance to this growth inhibitor. This cross-resistance did not appear to be due to a lack of uptake or retention of the respective inhibitors, since the mutants took up and retained more 3H-cAMP and 3H-RA than wild type cells, suggesting that the dual resistance was not due to an amplification of P-glycoprotein. The mutation confering cAMP-resistance did not appear to involve cyclic AMP-dependent protein kinase, since both catalytic activity and the amount of cAMP protein binding was similar in wild type and mutants. Thus, the mutation must be beyond the interaction of cAMP with cAMP-dependent protein kinase. We have previously reported that RA induces protein kinase C in B16 melanoma cells (Niles and Loewy: Cancer Res. 49:4483-4487, 1989). Therefore, we measured the ability of RA to induce protein kinase C in the cyclic AMP-resistant mutants. We found an inverse correlation between RA-induced protein kinase C activity and growth inhibition in these mutants. The data reported here suggest that cyclic AMP regulates some step in the RA signal transduction pathway.
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PMID:B16 mouse melanoma cells selected for resistance to cyclic AMP-mediated growth inhibition are cross-resistant to retinoic acid-induced growth inhibition. 164 60

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel requires both phosphorylation of its R domain and the presence of nucleoside triphosphates for activation. Our previous work suggested that hydrolysis of nucleoside triphosphates may be required to support channel activity. However, recent studies have suggested that the nonhydrolyzable adenosine triphosphate analogue, 5'-adenylylimidodiphosphate (AMP-PNP), may support some Cl- channel activity in sweat gland duct epithelia in the presence of low ATP concentration and in Cl- channels associated with expression of the P-glycoprotein multidrug resistance transporter. To examine the effect of AMP-PNP, we applied it to the cytosolic surface of phosphorylated CFTR Cl- channels contained in excised, cell-free patches of membrane. We found that preparations of 10 mM AMP-PNP opened phosphorylated CFTR Cl- channels. However, this effect was due to contaminating ATP: high-pressure liquid chromatography analysis of AMP-PNP demonstrated that 10 mM AMP-PNP could contain up to 50 microM ATP, which could account for the observed stimulation of CFTR Cl- channel activity. When contaminating ATP was hydrolyzed with hexokinase, AMP-PNP was unable to support CFTR channel activity. AMP-PNP (10 mM) also failed to attenuate or potentiate the current induced by 0.3 mM ATP. These results suggest that AMP-PNP has no direct effect on CFTR Cl- channels.
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PMID:5'-Adenylylimidodiphosphate does not activate CFTR chloride channels in cell-free patches of membrane. 768 26

Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.
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PMID:Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein. 790 31

Cytochemical screening for a panel of enzymes revealed increased 5' nucleotidase (5'NT) expression in 3 of 3 P-glycoprotein 170 (Pgp170)-positive multidrug-resistant (MDR) variants of the murine EL4 T-lymphoma cell line (EL4/ADM, ER2 and ER13). Electron microscopic localization established the presence of the membrane-bound ecto-form of the enzyme. Nine other murine, human and Chinese hamster cell lines and their MDR variants were tested for ecto-5'NT. Of these, 4 MDR variants (human cell lines MCF7A6, MCF7A2, HeLaJ2C and the murine cell line L1210A) showed increased expression of ecto-5'NT, when compared with their parental cell lines. The findings with cells of human origin were confirmed by immunofluorescent localization with a specific monoclonal antibody (MAb) (27.2) against the human ecto-5'NT. All MDR cell lines with elevated ecto-5'NT expression were generated by doxorubicin treatment. These cells were more sensitive than their parental cell lines to AMP at concentrations of 1.5-3.0 mM, confirming that the expressed ecto-5'NT was biologically active. The parental and MDR cells did not differ, in general, in their sensitivity to adenosine. An inhibitor of ecto-5'NT, alpha,beta-methyleneadenosine 5'-diphosphate, completely reversed the resistance of the EL4/ADM cell line to doxorubicin. The possibility exists of a functional relationship between the ecto-5'NT molecule and the members of the ATP-binding cassette transporter superfamily, important components of MDR, in some cell types.
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PMID:Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin. 792 9

The regulation of Cl- and cation conductances by the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) was characterized in isolated zymogen granules (ZG) from pancreatic acinar cells. ZG were purified from rat pancreas homogenate by Percoll gradient centrifugation. Cl- conductance was assayed by suspending ZG in isotonic KCl buffer and measuring osmotic lysis induced by maximal permeabilization of ZG membranes (ZGM) for K+ with the K+ ionophore valinomycin (Val). This resulted in influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances ZG (pHi approximately 6) were suspended in pH 7 buffered isotonic monovalent cation acetate salts. The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with the protonophore carbonyl cyanide p-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential driven influx of monovalent cations through endogenous channels and non-ionic diffusion of the counterion acetate. In the absence of Val, ZG were stable in KCl buffer up to 2 h. AMP-PCP enhanced osmotic lysis approximately 4-fold compared to control, due to activation of Cl- conductance by AMP-PCP and K+ influx through an AMP-PCP-insensitive nonselective cation pathway, which could be blocked by 0.1 mM Ba2+, 0.5 mM quinine, or 0.2 mM flufenamate. In addition, a K+ and Rb+ selective cation conductance was found which was completely blocked by 0.5 mM AMP-PCP or 0.5 mM quinine. AMP-PCP induced Cl- conductance was strongly inhibited by two monoclonal antibodies against MDR1 P-glycoprotein (JSB-1 and C219; 5-10 micrograms/ml), but not by a monoclonal antibody against the cystic fibrosis transmembrane conductance regulator (M3A7; 5 micrograms/ml) or by mouse IgG. The AMP-PCP insensitive nonselective cation conductance was not blocked by monoclonal antibodies against MDR1 P-glycoprotein (MDR1). Immunoblot studies of ZG membranes revealed the presence of a major immunoreactive protein band of approximately 65 kDa with both monoclonal antibodies against MDR1, but no protein of the approximate size of MDR1 (approximately 170 kDa) was detected. We propose that the Cl- channel or a regulator of the channel, that is activated by the non-hydrolyzable ATP analog AMP-PCP in ZG membranes, is a member of the ATP binding cassette superfamily of transporters and may have homology to MDR1 P-glycoprotein.
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PMID:Monoclonal antibodies against MDR1 P-glycoprotein inhibit chloride conductance and label a 65-kDa protein in pancreatic zymogen granule membranes. 792 2

A multidrug-resistant Chinese hamster ovary cell line (CR1R12) was obtained which constitutively expresses P-glycoprotein, up to 32% by weight of plasma membrane protein. CR1R12 plasma membranes had high, drug-activated ATPase activity referable to P-glycoprotein. The specific ATPase activity in the presence of verapamil was calculated to be approximately 9 mumol/min/mg (identical to 21 s-1) at 37 degrees C, pH 7.4. KM ATP was 1.4 mM, and ADP and 5'-adenylyl imidodiphosphate were competitive inhibitors with Ki values 0.35 and 0.44 mM, respectively. 2'-dATP was a good substrate, GTP and ITP were real but poor substrates, and ADP and AMP were not hydrolyzed. Optimal pH for ATP hydrolysis was 7.3. MgATP was the preferred substrate, and CaATP was hydrolyzed very weakly. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) covalently labeled the P-glycoprotein, and incorporation of 1.1 mol of NBD-Cl/mol of P-glycoprotein gave 100% inactivation. ATP protected against NBD-Cl inactivation. N-Ethylmaleimide was a potent inhibitor in the absence of ATP, and in its presence significant protection from inhibition could be achieved. Vanadate and fluoroaluminate were also strong inhibitors. The plasma membranes from CR1R12 cells should provide material for purification and reconstitution of P-glycoprotein and for screening of potential "multidrug-reversal" reagents by enzymic assay.
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PMID:Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein. 809 47

The Plasmodium falciparum P-glycoprotein homologue 1 (PGH1) is structurally similar to several members of the ATP-binding cassette (ABC) superfamily of membrane transporters. We have examined whether the nucleotide binding domains predicted from the deduced amino sequence are functional by photoaffinity labeling of purified parasite digestive vacuoles with the analogue 8-azido-alpha-[32P]ATP (8-N3-ATP). This reagent labels a 160-kDa protein in vacuoles from both a chloroquine sensitive and a chloroquine-resistant parasite isolate. The 160-kDa protein could be immunoprecipitated with affinity-purified antibodies against the P. falciparum P-glycoprotein homologue (PGH1). Inhibition of photoaffinity labeling of PGH1 could be achieved with ATP, ADP, GTP and GDP but not with AMP or GMP. In order to map the 8-N3-ATP binding sites on PGH1, photoaffinity-labeled PGH1 was digested with trypsin and immunoprecipitated with site-specific antibodies. Taken together, these results indicate that 8-N3-ATP specifically labels PGH1 and that one binding site resides within the amino terminal half of the molecule. This supports the contention that PGH1 is involved in a nucleotide-regulated transport function across the membrane of the digestive vacuole.
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PMID:Nucleotide binding properties of a P-glycoprotein homologue from Plasmodium falciparum. 809 60

8-Chloro-cyclic AMP (8-Cl-cAMP) produces growth-inhibitory and differentiating activity in the promyelocytic leukemia cell line HL-60. Adriamycin (ADR)-resistant HL-60 (HL-60/AR) cells exhibit the multidrug-resistant phenotype but do not express the mdr1 gene product P-glycoprotein. To explore potential signaling processes that may be involved in this atypical form of drug resistance, 8-Cl-cAMP was used as a modulator of the cAMP second messenger signal transduction pathway. Treatment for 48 hr with a 10% inhibitory concentration of 8-Cl-cAMP potentiated ADR cytotoxicity 14-fold in HL-60/AR cells but not in the parental cell line. 8-Cl-cAMP was stable to hydrolysis in the medium after 48 hr and was present intracellularly predominantly as phosphorylated metabolites (70%) and the parent compound (30%). No difference occurred in ADR accumulation in HL-60/AR cells after treatment with 8-Cl-cAMP. Accompanying the 8-Cl-cAMP-mediated increase in ADR cytotoxicity in HL-60/AR cells was a reduction in the cytosolic type I cAMP-dependent protein kinase (PKA) and disappearance of the nuclear PKA holoenzyme. Coincident with these changes in drug-resistant cells was a marked reduction in the DNA-binding activity of the cAMP response element-binding protein to levels equivalent to those in sensitive cells. This effect appears to result from reduced phosphorylation of the cAMP response element-binding protein. These results suggest that the potentiation by 8-Cl-cAMP of ADR cytotoxicity in HL-60/AR cells occurs through down-regulation of nuclear type I PKA and cAMP response element-binding factors whose activities are regulated by PKA.
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PMID:Reversal of resistance to adriamycin by 8-chloro-cyclic AMP in adriamycin-resistant HL-60 leukemia cells is associated with reduction of type I cyclic AMP-dependent protein kinase and cyclic AMP response element-binding protein DNA-binding activities. 838 2


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