Simultaneous Expression of Two P-Glycoprotein Genes in Drug-Sensitive Chinese Hamster Ovary Cells

Endicott, J.A.; Juranka, Peter F.; Sarangi, Farida; Gerlach, James H.; Deuchars, Kathryn L.; Ling, Victor

doi:10.1128/mcb.7.11.4075-4081.1987

Cited by 5 publications

(5 citation statements)

References 24 publications

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“…Photoaffinity labeling of mutant or wild-type MDR1 proteins with [a-32P]8-azido ATP was carried out as follows. A 60-,ug sample of purified membranes (1 mg/ml) was suspended in reaction buffer (50 mM Tris [pH 7.5], 50 mM KCl, 2 mM magnesium acetate, 0.1 mM dithiothreitol, 6% glycerol) in the presence of 10 ,.aCi of [a-32P]8-azido ATP (600 ,uCi/ml; specific activity, 7 Ci/mmol; ICN Biomedicals, Irvine, Calif.) and UV irradiated on ice for 10 min with a 15-W G15T8 germicidal lamp (General Electric Co., Schenectady, N.Y.) at a distance of 5 cm (31). Photoaffinity-labeled MDR1 proteins were immunoprecipitated with the anti-P glycoprotein monoclonal antibody C219 and analyzed by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Discrete Mutations Introduced in the Predicted Nucleotide-Binding Sites of the mdr1 Gene Abolish Its Ability To Confer Multidrug Resistance

Azzaria

Schurr

Gros

1989

Molecular and Cellular Biology

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In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

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Section: Methodsmentioning

confidence: 99%

Discrete Mutations Introduced in the Predicted Nucleotide-Binding Sites of the mdr1 Gene Abolish Its Ability To Confer Multidrug Resistance

Azzaria

Schurr

Gros

1989

Molecular and Cellular Biology

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“…The mammalian mdr gene family is composed of two members in humans and three members in rodents (34). Full-length cDNA clones have been obtained for the two human mdr genes (4,48), while partial cDNA (11) and genomic clones (34) have been described for the hamster pgp family. In the mouse, full-length cDNA clones for mdrl (21) and mdr2 (22) and partial cDNA clones for mdr3 (9,25) have been reported, and the complete genomic structure of the mdrl gene has been elucidated (37).…”

mentioning

confidence: 99%

Two Members of the Mouse mdr Gene Family Confer Multidrug Resistance with Overlapping but Distinct Drug Specificities

Devault

Gros

1990

Molecular and Cellular Biology

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We report the cloning and functional analysis of a complete clone for the third member of the mouse mdr gene family, mdr3. Nucleotide and predicted amino acid sequence analyses showed that the three mouse mdr genes encode highly homologous membrane glycoproteins, which share the same length (1,276 residues), the same predicted functional domains, and overall structural arrangement. Regions of divergence among the three proteins are concentrated in discrete segments of the predicted polypeptides. Sequence comparison indicated that the three mouse mdr genes were created from a common ancestor by two independent gene duplication events, the most recent one producing mdr1 and mdr3. When transfected and overexpressed in otherwise drug-sensitive cells, the mdr3 gene, like mdr1 and unlike mdr2, conferred multidrug resistance to these cells. In independently derived transfected cell clones expressing similar amounts of either MDR1 or MDR3 protein, the drug resistance profile conferred by mdr3 was distinct from that conferred by mdr1. Cells transfected with and expressing MDR1 showed a marked 7- to 10-fold preferential resistance to colchicine and Adriamycin compared with cells expressing equivalent amounts of MDR3. Conversely, cells transfected with and expressing MDR3 showed a two- to threefold preferential resistance to actinomycin D over their cellular counterpart expressing MDR1. These results suggest that MDR1 and MDR3 are membrane-associated efflux pumps which, in multidrug-resistant cells and perhaps normal tissues, have overlapping but distinct substrate specificities.

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“…Although active transport was known, it was largely studied in bacteria and unrecognized regarding a role in drug metabolism. Although a phenomenon known as multiple drug resistance was known to be operative in cancer cells and relevant to therapy (Endicott et al, 1987), the significance of these proteins in drug metabolism was not appreciated until later (Schuetz et al 1995).…”

Section: Changes and Progressmentioning

confidence: 99%

Drug Metabolism: A Half-Century Plus of Progress, Continued Needs, and New Opportunities

Guengerich

2022

Drug Metab Dispos

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The systematic study of drug metabolism began in the 19 th Century but most of what we know now has been learned in the last 50 years. Drug metabolism continues to play a critical role in pharmaceutical development and clinical practice, as well as contributing to toxicology, chemical carcinogenesis, endocrinology, and drug abuse. The importance of the field will continue, but its nature will continue to develop with changes in analytical chemistry, structural biology, and artificial intelligence. Challenges and opportunities include toxicology, defining roles of genetic variations, and application to clinical issues. Although the focus of this MiniReview is cytochrome P450, the same principles apply to other enzymes and transporters involved in drug metabolism.

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Simultaneous Expression of Two P-Glycoprotein Genes in Drug-Sensitive Chinese Hamster Ovary Cells

Cited by 5 publications

References 24 publications

Discrete Mutations Introduced in the Predicted Nucleotide-Binding Sites of the mdr1 Gene Abolish Its Ability To Confer Multidrug Resistance

Discrete Mutations Introduced in the Predicted Nucleotide-Binding Sites of the mdr1 Gene Abolish Its Ability To Confer Multidrug Resistance

Two Members of the Mouse mdr Gene Family Confer Multidrug Resistance with Overlapping but Distinct Drug Specificities

Drug Metabolism: A Half-Century Plus of Progress, Continued Needs, and New Opportunities

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