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)

MRP is a recently described ATP-binding cassette transporter that confers cellular resistance to natural product cytotoxic drugs. To examine the biochemical activity and cellular physiology of this transporter, we isolated the murine MRP homologue and analyzed its in vitro substrate specificity. Murine MRP transcript is widely expressed in tissues and encodes a protein of 1528 amino acids that is 88% identical to its human homologue. Hydropathy analysis indicated that murine and human MRP, the yeast cadmium resistance transporter and the sulfonylurea receptor share a conserved topology distinguished from P-glycoprotein and the cystic fibrosis conductance regulator by an N-terminal hydrophobic region that contains several potential transmembrane domains. Drug uptake assays performed with membrane vesicles prepared from NIH3T3 cells transfected with a murine MRP expression vector revealed ATP-dependent transport for the natural product cytotoxic drugs daunorubicin and vincristine, as well as for the glutathione S-conjugates leukotriene C4 and azidophenacyl-S-glutathione. Drug transport was osmotically sensitive and saturable with regard to drug and ATP concentrations, with K(m) values of 19 microM, 19 microM, 26 nM, 17 microM, and 77 microM for daunorubicin, vincristine, leukotriene C4, APA-SG, and ATP, respectively. Consistent with broad substrate specificity, the drug glutathione conjugate APA-SG, oxidized glutathione, the LTD4 antagonist MK571, arsenate, and genistein were competitive inhibitors of daunorubicin transport, with Ki values of 32 microM, 25 microM, 1.9 microM, 108 microM, and 23 microM, respectively. This study demonstrates that the substrate specificity of murine MRP is quite broad and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression pattern of murine MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that it represents a widespread and versatile cellular defense mechanism.
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PMID:Structure and in vitro substrate specificity of the murine multidrug resistance-associated protein. 954 10

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated C1(-) channel. Malfunction of CFTR causes cystic fibrosis (CF). CFTR belongs to an ATP-binding cassette (ABC) transporter superfamily which includes P-glycoprotein (Pgp), the molecule that is responsible for multidrug resistance in cancer cells. P-glycoprotein molecules have been suggested to have more than one topology and function. In this study, we analysed the early stages of membrane insertion, processing, and topology of human CFTR using rabbit reticulocyte lysate and wheat germ extract translation systems supplemented with canine pancreatic microsomal membranes. Our results suggest that CFTR contains an uncleavable signal sequence and its membrane targeting and insertion may depend on the signal recognition particle (SRP) and SRP receptor. The topology of CFTR in microsomal membranes is the same as the one predicted based on hydropathy plot analysis. These results, together with our previous findings on Pgp, indicate that (1) the topologies of mammalian ABC transporters can be dissected and studied using protein fusion chimeras in a cell-tree system; and (2) the membrane targeting and insertion of CFTR and Pgp may take the same pathway, i.e., the SRP-dependent pathway, but the membrane folding mechanism of these two proteins in microsomal membranes is probably different.
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PMID:Membrane insertion, processing, and topology of cystic fibrosis transmembrane conductance regulator (CFTR) in microsomal membranes. 914 60

ABC transporters are a large superfamily of integral membrane proteins involved inATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multidrug resistance (P-glycoprotein, MRP). Most ABC transporters are predicted to consist of four domains, two membrane-spanning domains and two cytoplasmic domains. The latter contain conserved nucleotide-binding motifs. Attempts to determine the structure of ABC transporters and of their separate domains are in progress but have not yet been successful. To aid structure determination and possibly learn more about the domain boundaries, we set out to model nucleotide-binding domains (NBDs) of ABC transporters based on a known structure. Previous attempts to predict the 3D structure of NBDs were based solely on sequence similarity with known nucleotide-binding folds. We have analyzed the sequences of a number of nucleotide-binding domains with the algorithm THREADER, developed by D.T. Jones, and a possible fold was found in the structure of aspartate aminotransferase. We present a model for the N-terminal NBD of CFTR, based on the large domain of the A chain of aspartate aminotransferase. The model is refined using multiple sequence alignment, secondary structure prediction, and 3D-1D profiles. Our model seems to be in good agreement with known properties of nucleotide-binding domains and has some appealing characteristics compared with the previous models.
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PMID:A model for the nucleotide-binding domains of ABC transporters based on the large domain of aspartate aminotransferase. 951 43

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl- and ATP. Similar to previously reported data for in vivo ATP current of CFTR-expressing cells, the reconstituted channels displayed competition between Cl- and ATP and had multiple conductance states in the presence of Cl- and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the "intracellular" side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.
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PMID:Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator. 953 Jan 12

Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.
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PMID:ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. 1079 28

The ABC superfamily of membrane transporters is one of the largest classes of proteins across all species and one of the most intensely researched. ABC proteins are involved in the trafficking of a diverse variety of biological molecules across cell membranes, with some members implicated in medical syndromes such as cystic fibrosis and multidrug resistance to anti-cancer drugs. In the absence of X-ray crystallographic data, structural information has come from spectroscopy, electron microscopy, secondary structure prediction algorithms and residue substitution, epitope labelling and cysteine cross-linking studies. These have generally supported a model for the topology of the transmembrane domains of ABC transporters in which a single aqueous pore is formed by a toroidal ring of 12 alpha helices, deployed in two arcs of six helices each. Although this so-called 6 + 6 helix model can be arranged in either mirror or rotational symmetry configurations, experimental data supports the former. In this review, we put forward arguments against both configurations of this 6 + 6 helix model, based on what is known generally about symmetry relationships in proteins. We relate these arguments to P-glycoprotein, in particular, and discuss alternative models for the structure of ABC transporters in the light of the most recent research.
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PMID:Symmetry and structure in P-glycoprotein and ABC transporters what goes around comes around. 1095 Nov 88

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K(+) channels [1-4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5-8]. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. The crystal structure of these domains has even been described recently [9, 10]. However, nothing is known of the function or organization of the 12 transmembrane segments. In the present study we adopted a range of strategies including live-cell fluorescence resonance energy transfer (FRET) microscopy, coimmunoprecipitation, and functional assays of various truncated and substituted, fluorescently-tagged molecules to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains - largely independently of any cytosolic region - formed a tight complex that was delivered to the plasma membrane. This cooperation between the two intact transmembrane domains was essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, was also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell.
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PMID:Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase. 1123 Nov 54

Macrolide antibiotics are strongly concentrated within host cells, a property that sustains their activity against intracellular pathogens and is likely responsible for the modulation of cell metabolism and function. There is extensive literature on the subject of macrolide-induced modulation of immune responses. Erythromycin A derivatives seem to display anti-inflammatory activity in vitro, in some animal models and in various clinical settings such as diffuse panbronchiolitis (DPB). The underlying mechanisms are not yet fully understood: inflammatory cytokine and oxidant production by phagocytes is down-regulated by these drugs, but other possible targets include bacterial virulence factors, bronchial and epithelial cells, etc. Also, a link has been suggested between the macrolide transmembrane carrier system and the P-glycoprotein family, which comprises MDR (multiple drug resistance) and CFTR (cystic fibrosis transmembrane conductance regulator), which are respectively involved in the chemotherapeutic resistance of cancer cells and in the genesis of cystic fibrosis.
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PMID:Immunomodulation by macrolide antibiotics. 1123 97

The most common mutation in cystic fibrosis (deletion of phenylalanine 508 (DeltaF508) in the cystic fibrosis conductance transmembrane regulator (CFTR) gene) causes defective synthesis of CFTR protein. To understand how this deletion interferes with protein folding, we made the equivalent deletion (DeltaY490) in P-glycoprotein (P-gp). A Cys-less P-gp with cysteines in transmembrane (TM) 4 or TM5 can be cross-linked with a cysteine in TM12. Deleting Tyr(490) in P-gp resulted in an inactive and defectively processed mutant in which no cross-linking between TM4 or TM5 and TM12 was detected. Expression of the DeltaY490 mutant in the presence of a chemical chaperone corrected the processing defect and yielded active P-gp mutants that could be cross-linked between TM4 or TM5 and TM12. Cross-linking between TM4 or TM5 and TM12 was also detected when residues (483)TIAENIRYG(491) in P-gp were replaced with residues (501)TIKENIIFG(509) from CFTR (P-gp/CFTR). Deleting Phe(508) in the P-gp/CFTR chimera, however, caused defective processing of the mutant protein and no detectable cross-linking between TM4 or TM5 and TM12. The processing defect was corrected with a chemical chaperone and yielded active P-gp/CFTR mutant proteins that could be cross-linked. These results show that deletion at residue 490 disrupts packing of the TM segments possibly by affecting interaction between the first nucleotide-binding domain (Tyr(490)) and the first cytoplasmic loop (Glu(184)).
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PMID:Introduction of the most common cystic fibrosis mutation (Delta F508) into human P-glycoprotein disrupts packing of the transmembrane segments. 1207 Jan 34

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.
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PMID:Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein. 1236 99


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