Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance - PubMed (original) (raw)
Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance
M Azzaria et al. Mol Cell Biol. 1989 Dec.
Abstract
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.
Similar articles
- Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities.
Devault A, Gros P. Devault A, et al. Mol Cell Biol. 1990 Apr;10(4):1652-63. doi: 10.1128/mcb.10.4.1652-1663.1990. Mol Cell Biol. 1990. PMID: 1969610 Free PMC article. - A single amino acid substitution strongly modulates the activity and substrate specificity of the mouse mdr1 and mdr3 drug efflux pumps.
Gros P, Dhir R, Croop J, Talbot F. Gros P, et al. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7289-93. doi: 10.1073/pnas.88.16.7289. Proc Natl Acad Sci U S A. 1991. PMID: 1678520 Free PMC article. - Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site.
Kajiji S, Talbot F, Grizzuti K, Van Dyke-Phillips V, Agresti M, Safa AR, Gros P. Kajiji S, et al. Biochemistry. 1993 Apr 27;32(16):4185-94. doi: 10.1021/bi00067a005. Biochemistry. 1993. PMID: 7682843 - ATP hydrolysis-dependent multidrug efflux transporter: MDR1/P-glycoprotein.
Kimura Y, Matsuo M, Takahashi K, Saeki T, Kioka N, Amachi T, Ueda K. Kimura Y, et al. Curr Drug Metab. 2004 Feb;5(1):1-10. doi: 10.2174/1389200043489090. Curr Drug Metab. 2004. PMID: 14965247 Review. - Molecular analysis of the multidrug transporter.
Germann UA. Germann UA. Cytotechnology. 1993;12(1-3):33-62. doi: 10.1007/BF00744657. Cytotechnology. 1993. PMID: 7765331 Review.
Cited by
- Mechanistic toxicology in light of genetic compensation.
Elizalde MJ, Gorelick DA. Elizalde MJ, et al. Toxicol Sci. 2023 Nov 6;197(2):115-20. doi: 10.1093/toxsci/kfad113. Online ahead of print. Toxicol Sci. 2023. PMID: 37941503 - P-glycoprotein: new insights into structure, physiological function, regulation and alterations in disease.
Ahmed Juvale II, Abdul Hamid AA, Abd Halim KB, Che Has AT. Ahmed Juvale II, et al. Heliyon. 2022 Jun 22;8(6):e09777. doi: 10.1016/j.heliyon.2022.e09777. eCollection 2022 Jun. Heliyon. 2022. PMID: 35789865 Free PMC article. Review. - Pharmaceutical Formulations with P-Glycoprotein Inhibitory Effect as Promising Approaches for Enhancing Oral Drug Absorption and Bioavailability.
Nguyen TT, Duong VA, Maeng HJ. Nguyen TT, et al. Pharmaceutics. 2021 Jul 20;13(7):1103. doi: 10.3390/pharmaceutics13071103. Pharmaceutics. 2021. PMID: 34371794 Free PMC article. Review. - The role of the degenerate nucleotide binding site in type I ABC exporters.
Stockner T, Gradisch R, Schmitt L. Stockner T, et al. FEBS Lett. 2020 Dec;594(23):3815-3838. doi: 10.1002/1873-3468.13997. Epub 2020 Nov 27. FEBS Lett. 2020. PMID: 33179257 Free PMC article. Review. - In vivo FRET analyses reveal a role of ATP hydrolysis-associated conformational changes in human P-glycoprotein.
Futamata R, Ogasawara F, Ichikawa T, Kodan A, Kimura Y, Kioka N, Ueda K. Futamata R, et al. J Biol Chem. 2020 Apr 10;295(15):5002-5011. doi: 10.1074/jbc.RA119.012042. Epub 2020 Feb 28. J Biol Chem. 2020. PMID: 32111736 Free PMC article.
References
- Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 - PubMed
- Annu Rev Biochem. 1989;58:137-71 - PubMed
- Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373-6 - PubMed
- Nature. 1970 Aug 15;227(5259):680-5 - PubMed
- Cancer Res. 1970 Apr;30(4):1174-84 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Molecular Biology Databases
Research Materials
Miscellaneous