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)

HL60 cells isolated for resistance to Adriamycin are multidrug resistant and defective in the cellular accumulation of drug. These cells do not however overexpress mdr1 and do not contain detectable levels of P-glycoprotein. In the present study we have prepared antisera against synthetic peptides that correspond to various sequence domains of P-glycoprotein and have examined by Western blot analysis the reactivity of these antisera with proteins contained in membranes of HL60/Adr cells. All antisera are highly reactive with a Mr 180,000 (p180) P-glycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine (HL60/Vinc). In contrast, of 13 antisera tested 12 do not react with any resistance-associated protein in the HL60/Adr isolate. One antiserum (ASP14) is however highly reactive with a Mr 190,000 protein (p190) contained in HL60/Adr membranes. This protein is not detected in drug-sensitive cells. ASP14 also reacts with proteins p195 and p50 contained in a second independent HL60/Adr isolate. Analysis of membrane subfractions shows that p190 is located primarily in the endoplasmic reticulum with only low levels contained in plasma membranes. Additional studies demonstrate that endoplasmic reticulum of HL60/Adr cells contain a major Mr 190,000 protein that is capable of binding the photoaffinity agent 8-azido[alpha-32P]ATP. p195 contained in a second HL60/Adr isolate is also labeled with 8-azido[alpha-32P]ATP. These results thus demonstrate that antiserum against a specific P-glycoprotein sequence detects a p190 (p195) resistance-associated membrane protein in two independent HL60/Adr isolates. p190 (p195) and P-glycoprotein thus contain a minor sequence homology and based on the specificity of ASP14 this occurs in a region which may be involved in nucleotide binding. Possibly this sequence is common to and essential for the functionality of proteins which contribute to resistance by reducing cellular drug levels.
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PMID:Mechanisms of multidrug resistance in HL60 cells: detection of resistance-associated proteins with antibodies against synthetic peptides that correspond to the deduced sequence of P-glycoprotein. 196 79

Monoclonal antibody against the Mr 22,000 calcium-binding protein (sorcin) from an adriamycin-resistant myelogenous leukemia cell line K562 (K562/ADM) was prepared and used as a probe to study the localization of sorcin in K562/ADM cells and the parental cell line, K562. Analysis of extracts from K562/ADM cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence image analysis showed that K562/ADM cells possessed abundant sorcin in the cytoplasm which was almost entirely absent from the drug-sensitive parental cell line, K562. Furthermore, immuno-electron microscopic studies revealed that sorcin was closely associated with free ribosomes, rough endoplasmic reticulum, mitochondria, microfilament bundles and perinuclear membranes. These observations provide the first clue that the Ca-binding protein, sorcin, may play an important role in the development of the multidrug resistance phenomenon, although the relationship between sorcin and P-glycoprotein is still unknown.
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PMID:Immunocytochemical identification and localization of the Mr 22,000 calcium-binding protein (sorcin) in an adriamycin-resistant myelogenous leukemia cell line. 256 83

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.
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PMID:Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells. 289 Jun 86

P-glycoprotein (Mdr1), a member of the ABC superfamily, is a pump able to transport several compounds across plasma membranes. It displays a high level of similarity with the MHC-linked transporters TAP1 and TAP2 which are involved in the delivery of immunogenic peptides across the endoplasmic reticulum. In the present study we analyze the P-glycoprotein's ability to interfere with the biosynthetic pathway of the MHC class I molecules. Our results show that P-glycoprotein is involved in the modulation of the MHC class I expression in multidrug-resistant tumor cell lines, COS1 cells transfected with mdr1 gene, and human T lymphocytes. Epitope screening evokes the possibility that P-glycoprotein induces a modulation of the different MHC class I forms expressed on the cell surface. We propose that P-glycoprotein is involved in the transport of antigenic protein fragments from the cytosol into the endoplasmic reticulum. The suggested mechanism could be physiologically relevant in tissues displaying a high Mdr1 activity, where this transporter could contribute to the regulation of locoregional immune responses.
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PMID:Cell surface expression of major histocompatibility class I antigens is modulated by P-glycoprotein transporter. 775 13

Transmembrane topology of polytopic integral membrane proteins is established during protein synthesis at the endoplasmic reticulum membrane. For some polytopic proteins, sequential and independent signal, stop transfer, and/or signal anchor sequences contained in the nascent chain direct this process. Here we define the topology of human P-glycoprotein (MDR1) through the first two transmembrane regions (TM1 and TM2, respectively) of the amino-terminal half of the protein. We show that unlike TM7 and TM8, which comprise homologous regions in the carboxyl half of the protein (Skach, W., Calayag, M. C., and Lingappa, V. (1993) J. Biol. Chem. 268, 6903-6908), TM1 and TM2 achieve the orientation predicted by conventional structural models. However, TM1 and TM2 appear to utilize a mechanism of biogenesis different in a key respect from that observed in multispanning proteins studied previously. TM1 and TM2, with their flanking regions, independently direct the topology observed for each of these sequences in the native protein. Each can interact with signal recognition particle to direct targetting to the endoplasmic reticulum, nascent chain translocation, and correct transmembrane orientation. Unlike the transmembrane regions of previously studied multispanning membrane proteins, neither TM1 nor TM2 alone is sufficient to integrate the chain into the membrane. However, when TM1 and TM2 are both present, as occurs in native MDR1, integration is achieved. These results suggest that cooperative interactions between TM1 and TM2 are necessary for chain integration and thus add a new complexity to the current view of polytopic integral membrane protein biogenesis.
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PMID:Amino-terminal assembly of human P-glycoprotein at the endoplasmic reticulum is directed by cooperative actions of two internal sequences. 790 Dec 9

Site-directed mutagenesis was used to investigate whether glycine residues in the predicted cytoplasmic loops play essential roles in the structure and function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 20 glycine residues were changed to valine, were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutation of Gly-251, -268, -269, or -781 yielded mutant proteins which were unable to confer drug resistance in transfected cells. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were improperly processed so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. The in vivo processing of mutants Gly-269 to Val and Gly-781 to Val was temperature-sensitive. When cells expressing these mutants were grown at a lower temperature (26 degrees C), the mature 170-kDa form of P-glycoprotein was the major product. Substitution of glycine with alanine at positions 251, 268, 269, or 781 yielded mutants with structural and functional characteristics similar to wild-type enzyme. Mutation of Gly-141, 187, 288, 812, or 830 to Val, altered the drug resistance profile conferred by P-glycoproteins expressed in transfected cells. All five mutations increased relative resistance to colchicine or adriamycin, without altering relative resistance to vinblastine. These results demonstrate that glycines located in the cytoplasmic loops play important roles in structure and function of P-glycoprotein.
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PMID:Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein. 790 26

The vectorial transport of xenobiotics across the hepatocyte is mediated by various transport and transfer proteins that differ in ligand specificity and function. The influx of xenobiotics from the blood across the sinusoidal membrane of the hepatocyte can occur through passive or active transport processes. Once in the cell, xenobiotics can be sequestered by intracellular transfer proteins that prevent refluxing of the chemical back through the sinusoidal membrane. Transfer proteins may also facilitate the localization of the xenobiotics within the cell to sites of metabolism (i.e., the endoplasmic reticulum) or elimination (i.e., the canalicular membrane). Intracellular transfer proteins include glutathione S-transferases, fatty acid-binding proteins, and 3 alpha-hydroxysteroid dehydrogenase. Intracellular nuclear transfer proteins have also been identified that facilitate the transfer of chemical carcinogens from the cytoplasm into the cell nucleus. Several active transport proteins exist on the canalicular membrane of the hepatocyte that mediate the efflux of chemicals from the cell into the biliary canaliculus. Xenobiotic efflux proteins include the multispecific organic anion transporter, that eliminates xenobiotics that have undergone conjugation with glutathione, glucuronic acid, and possibly sulfate; and, P-glycoprotein, an active transporter that actively effluxes a variety of structurally diverse xenobiotics. Induction of P-glycoprotein by the amplification of its gene has been identified as a major cause of resistance of tumor cells to the toxicity of a variety of anti-cancer drugs. The hepatic induction of P-glycoprotein may also contribute to acquired resistance of organisms to environmental toxicants. Continued elucidation of xenobiotic transport and transfer processes at the cellular levels will significantly advance our understanding of processes involved in xenobiotic toxicity and acquired resistance to chemical toxicity.
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PMID:Hepatic vectorial transport of xenobiotics. 790 59

Understanding how the multidrug resistance phenotype is manifest in human cancer cells will require insight into the mechanism of assembly, transmembrane topology, and intracellular trafficking of human P-glycoprotein (MDR1). Previously, we showed that MDR1 amino terminus biogenesis occurred through an unexpected interaction between novel topogenic sequence subtypes and that transmembrane topology of corresponding amino and carboxy halves was not equivalent. We now investigate topology and topogenic activities of the third and fourth transmembrane regions (TM3 and TM4) of human MDR1 using protease protection of defined reporter epitopes expressed in Xenopus laevis oocytes. As was previously observed for TM1 and TM2, determinants in TM3 and TM4 exhibited cooperativity in directing proper assembly and transmembrane orientation. The signal sequence encompassing TM3 required residues from TM4 to reinitiate translocation of the MDR1 chain into the endoplasmic reticulum (ER) lumen. Remaining residues from TM4 terminated translocation and established a polytopic transmembrane topology in which TM3 and TM4 both spanned the membrane in the orientation predicted by hydropathy-based models. Remarkably, when translocating sequentially into the ER lumen, neither TM4 alone nor TM4 together with TM3 efficiently terminated translocation. Thus, MDR1 biogenesis required both the presence of these sequences and their proper orientation with respect to the ER translocation apparatus. This conclusion was supported by experiments in which TM3 and TM4 topology was reproduced in a defined chimeric protein which mimicked native MDR1 presentation. These additional variations on simple themes of protein topogenesis utilized by MDR1 demonstrate that events of complex protein biogenesis may be dissected and studied using protein chimeras with defined translocation properties.
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PMID:Transmembrane orientation and topogenesis of the third and fourth membrane-spanning regions of human P-glycoprotein (MDR1). 791 95

Mutation of amino acids located within or immediately NH2-terminal to transmembrane segment 7 of human P-glycoprotein abolished the ability of the protein to confer resistance to cytotoxic drugs. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein, and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were processed improperly, so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. Processing of the 150-kDa P-glycoprotein to the 170-kDa mature form of the enzyme for all of the mutants, except Glu707-->Ala and Tyr710-->Ala, was dramatically increased when the cells were grown at 26 degrees C. At the lower growth temperature, the mature protein was targeted to the plasma membrane, and drug efflux activity was restored. We also analyzed the mutants for possible molecular interactions that may contribute to their intracellular retention. We found that core-glycosylated forms of the wild-type and mutant P-glycoproteins were associated with the molecular chaperone calnexin. Only wild-type enzyme, however, was able to escape association with calnexin and be targeted to the plasma membrane. Prolonged association of the mutants with calnexin may be due to misfolding of the protein as evidenced by their relative short half-life of about 3 h, compared with 50 h for the wild-type enzyme. These results suggest that calnexin contributes to a quality control mechanism to retain misfolded forms of P-glycoprotein in the endoplasmic reticulum.
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PMID:Prolonged association of temperature-sensitive mutants of human P-glycoprotein with calnexin during biogenesis. 796 19

In an effort to define clearly the basis of non-P-glycoprotein multidrug resistance in HL60/ADR cells, we have analyzed expression of MRP mRNA levels and the MRP-encoded protein in resistant cells and also in resistant cells that have undergone a reversion to drug sensitivity. The results demonstrate that an MRP cDNA containing 5'-end coding sequences reacts with a 6-kb RNA, which is overexpressed in the resistant isolate. As resistant cells revert to drug sensitivity there is essentially a complete loss of the 6-kb RNA. Southern blot analysis indicates that the MRP gene is amplified compared to the copy number found in sensitive cells. Revertant cells no longer contain amplified MRP sequences. Western blot analysis has been conducted using an antibody prepared against the carboxyl terminus (15 amino acids) of the deduced sequence of the MRP-encoded protein. The antibody is reactive with a 190-kDa protein (P190) and with two closely migrating proteins of 65 and 70 kDa (P70), which are overexpressed in plasma membranes and endoplasmic reticulum of resistant cells. Both proteins are greatly reduced in revertant cells. Growth of cells in the presence of tunicamycin demonstrates that both P190 and P70 are glycosylated, with the deglycosylated forms migrating in polyacrylamide gels as proteins of 165 kDa and 45 kDa, respectively. Additional antisera have also been prepared against sequence domains contained in the C-terminal region of P190. These antisera are reactive with both P190 and P70. Antisera directed against sequences of the amino terminal region of P190 do not react with P70.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of MRP gene expression and function in HL60 cells isolated for resistance to adriamycin. 799 83


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