scholarly journals Functional Expression of Multidrug Resistance Protein 4 MRP4/ABCC4

2019 ◽  
Vol 24 (10) ◽  
pp. 1000-1008 ◽  
Author(s):  
David Hardy ◽  
Roslyn M. Bill ◽  
Anass Jawhari ◽  
Alice J. Rothnie
Keyword(s):  
Multidrug Resistance ◽  
Membrane Proteins ◽  
Transmembrane Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Functional Expression ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Multidrug Resistance Protein ◽  
Resistance Protein

To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

Functional Expression of the Multidrug Resistance Protein 1 in Microglia

2003 ◽  
Vol 307 (1) ◽  
pp. 282-290 ◽  
Author(s):  
Shannon Dallas ◽  
Xiaoping Zhu ◽  
Sylvain Baruchel ◽  
Lyanne Schlichter ◽  
Reina Bendayan
Keyword(s):  
Multidrug Resistance ◽  
Functional Expression ◽  
Multidrug Resistance Protein ◽  
Multidrug Resistance Protein 1 ◽  
Resistance Protein

scholarly journals Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520

1994 ◽  
Vol 14 (1) ◽  
pp. 277-286
Author(s):  
M Raymond ◽  
S Ruetz ◽  
D Y Thomas ◽  
P Gros
Keyword(s):  
Multidrug Resistance ◽  
Mammalian Cells ◽  
Drug Transport ◽  
Function Analysis ◽  
Expression System ◽  
Functional Expression ◽  
Yeast Cells ◽  
Mutant Form ◽  
Cellular Resistance ◽  
P Glycoprotein

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

Multidrug Resistance Protein 4 (MRP4/ABCC4): A Suspected Efflux Transporter for Human’s Platelet Activation

2021 ◽  
Vol 28 ◽  
Author(s):  
Ioannis Angelis ◽  
Vassilios Moussis ◽  
Demokritos C. Tsoukatos ◽  
Vassilios Tsikaris
Keyword(s):  
Multidrug Resistance ◽  
Platelet Activation ◽  
Transmembrane Protein ◽  
Main Role ◽  
Multidrug Resistance Protein ◽  
Efflux Transporter ◽  
Normal Platelet ◽  
Platelet Receptor ◽  
Resistance Protein

: The main role of platelets is to contribute to hemostasis. However, under pathophysiological conditions, platelet activation may lead to thrombotic events of cardiovascular diseases. Thus, anti-thrombotic treatment is important in patients with cardiovascular disease. This review focuses on a platelet receptor, a transmembrane protein, the Multidrug Resistance Protein 4, MRP4, which contributes to platelet activation by extruding endogenous molecules responsible for their activation and accumulation. The regulation of the intracellular concentration levels of these molecules by MRP4 turned to make the protein suspicious and, at the same time, an interesting regulatory factor of normal platelet function. Especially, the possible role of MRP4 in the excretion of xenobiotic and antiplatelet drugs such as aspirin is discussed, thus imparting platelet aspirin tolerance and correlating the protein with the ineffectiveness of aspirin antiplatelet therapy. Based on the above, this review finally underlines that the development of a highly selective and targeted strategy for platelet MRP4 inhibition will also lead to inhibition of platelet activation and accumulation.

Functional Expression of Multidrug Resistance Protein 1 inPichia pastoris†

Biochemistry ◽  
2001 ◽  
Vol 40 (28) ◽  
pp. 8307-8316 ◽  
Author(s):  
Jie Cai ◽  
Roni Daoud ◽  
Elias Georges ◽  
Philippe Gros
Keyword(s):  
Multidrug Resistance ◽  
Functional Expression ◽  
Multidrug Resistance Protein ◽  
Multidrug Resistance Protein 1 ◽  
Resistance Protein

scholarly journals Transport of leukotriene C4 by a cysteine-less multidrug resistance protein 1 (MRP1)

2003 ◽  
Vol 370 (1) ◽  
pp. 357-360 ◽  
Author(s):  
Sung H. LEE ◽  
Guillermo A. ALTENBERG
Keyword(s):  
Multidrug Resistance ◽  
Drug Transport ◽  
Transmembrane Domain ◽  
Low Cost ◽  
Expression System ◽  
Multidrug Resistance Protein ◽  
Leukotriene C4 ◽  
Multidrug Resistance Protein 1 ◽  
Yeast Expression System ◽  
Resistance Protein

Overexpression of multidrug resistance protein 1 (MRP1), an ATP-binding cassette protein, causes multidrug resistance. We developed a functional cysteine-less version of MRP1 that provides a framework for detailed biochemical and biophysical studies. The 18 Cys residues of a truncated MRP1 (tMRP1; lacking the first multispanning transmembrane domain) were replaced with Ala to generate Cys-less tMRP1 (CL tMRP1). CL tMRP1 expressed in Saccharomyces cerevisiae membranes displayed high-affinity ATP-dependent transport of the MRP1 substrate leukotriene C4. Compared with full-length MRP1, the Km for leukotriene C4 transport by CL tMRP1 was increased 3-fold, while Vmax was not affected. Thus a functional CL tMRP1 can be expressed using a low-cost and rapid-generation yeast expression system. This Cys-less protein can be used for biochemical, spectroscopic and structural studies to elucidate the mechanism of drug transport by MRP1.

scholarly journals Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4 (ATP-binding cassette C4)

2003 ◽  
Vol 371 (2) ◽  
pp. 361-367 ◽  
Author(s):  
Noam ZELCER ◽  
Glen REID ◽  
Peter WIELINGA ◽  
Annemieke KUIL ◽  
Ingrid van der HEIJDEN ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Bile Acid ◽  
Bile Acids ◽  
Mammalian Cells ◽  
Physiological Role ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
Multidrug Resistance Protein ◽  
Resistance Protein ◽  
Dependent Transport

Human multidrug-resistance protein (MRP) 4 transports cyclic nucleotides and when overproduced in mammalian cells mediates resistance to some nucleoside analogues. Recently, it has been shown that Mrp4 is induced in the livers of Fxr(-/-) mice, which have increased levels of serum bile acids. Since MRP4, like MRP1–3, also mediates transport of a model steroid conjugate substrate, oestradiol 17-β-d-glucuronide (E217βG), we tested whether MRP4 may be involved in the transport of steroid and bile acid conjugates. Bile salts, especially sulphated derivatives, and cholestatic oestrogens inhibited the MRP4-mediated transport of E217βG. Inhibition by oestradiol 3,17-disulphate and taurolithocholate 3-sulphate was competitive, suggesting that these compounds are MRP4 substrates. Furthermore, we found that MRP4 transports dehydroepiandrosterone 3-sulphate (DHEAS), the most abundant circulating steroid in humans, which is made in the adrenal gland. The ATP-dependent transport of DHEAS by MRP4 showed saturable kinetics with Km and Vmax values of 2μM and 45pmol/mg per min, respectively (at 27°C). We further studied the possible involvement of other members of the MRP family of transporters in the transport of DHEAS. We found that MRP1 transports DHEAS in a glutathione-dependent manner and exhibits Km and Vmax values of 5μM and 73pmol/mg per min, respectively (at 27°C). No transport of DHEAS was observed in membrane vesicles containing MRP2 or MRP3. Our findings suggest a physiological role for MRP1 and MRP4 in DHEAS transport and an involvement of MRP4 in transport of conjugated steroids and bile acids.

scholarly journals Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520.

1994 ◽  
Vol 14 (1) ◽  
pp. 277-286 ◽  
Author(s):  
M Raymond ◽  
S Ruetz ◽  
D Y Thomas ◽  
P Gros
Keyword(s):  
Multidrug Resistance ◽  
Mammalian Cells ◽  
Drug Transport ◽  
Function Analysis ◽  
Expression System ◽  
Functional Expression ◽  
Yeast Cells ◽  
Mutant Form ◽  
Cellular Resistance ◽  
P Glycoprotein

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

scholarly journals Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal

2000 ◽  
Vol 350 (2) ◽  
pp. 555-561 ◽  
Author(s):  
Johan RENES ◽  
Elisabeth E. G. DE VRIES ◽  
Guido J. E. J. HOOIVELD ◽  
Inge KRIKKEN ◽  
Peter L. M. JANSEN ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Multidrug Resistance ◽  
Cell Lines ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
Lipid Peroxidation Product ◽  
Multidrug Resistance Protein ◽  
Peroxidation Product ◽  
Resistance Protein

4-Hydroxynonenal (4HNE) is the most prevalent toxic lipid peroxidation product formed during oxidative stress. It exerts its cytotoxicity mainly by the modification of intracellular proteins. The detection of 4HNE-modified proteins in several degenerative disorders suggests a role for 4HNE in the onset of these diseases. Efficient protection mechanisms are required to prevent the intracellular accumulation of 4HNE. The toxicity of 4HNE was tested with the small cell lung cancer cell lines GLC4 and the multidrug-resistance-protein (MRP1)-overexpressing counterpart GLC4/Adr. In the presence of the MRP1 inhibitor MK571 or the GSH-depleting agent buthionine sulphoximine, both cell lines became more sensitive and showed decreased survival. Transport experiments were performed with the 3H-labelled glutathione S-conjugate of 4HNE ([3H]GS-4HNE) with membrane vesicles from GLC4-derived cell lines with different expression levels of MRP1. [3H]GS-4HNE was taken up in an ATP-dependent manner and the transport rate was dependent on the amount of MRP1. The MRP1 inhibitor MK571 decreased [3H]GS-4HNE uptake. MRP1-specific [3H]GS-4HNE transport was demonstrated with membrane vesicles from High Five insect cells overexpressing recombinant MRP1. Kinetic experiments showed an apparent Km of 1.6±0.21µM (mean±S.D.) for MRP1-mediated [3H]GS-4HNE transport. In conclusion, MRP1 has a role in the protection against 4HNE toxicity and GS-4HNE is a novel MRP1 substrate. MRP1, together with GSH, is hypothesized to have a role in the defence against oxidative stress.

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