Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Aug 15;88(16):7289–7293. doi: 10.1073/pnas.88.16.7289

A single amino acid substitution strongly modulates the activity and substrate specificity of the mouse mdr1 and mdr3 drug efflux pumps.

P Gros 1, R Dhir 1, J Croop 1, F Talbot 1
PMCID: PMC52280  PMID: 1678520

Abstract

Specific protein domains and amino acids responsible for the apparent capacity of P-glycoprotein (mdr) to recognize and transport a large group of structurally unrelated drugs have not been identified. We have introduced a single Ser----Phe substitution within the predicted TM11 domain of mdr1 (position 941) and mdr3 (position 939) and analyzed the effect of these substitutions on the drug-resistance profiles of these two proteins. Mutations at this residue drastically altered the overall degree of drug resistance conveyed by mdr1 and mdr3. The modulating effect of this mutation on mdr1 and mdr3 varied for the drugs tested: it was very strong for colchicine and adriamycin and moderate for vinblastine. For mdr1, the Ser941----Phe941 substitution produced a unique mutant protein that retained the capacity to confer vinblastine resistance but lost the ability to confer adriamycin and colchicine resistance. These results strongly suggest that the predicted TM11 domain of proteins encoded by mdr and mdr-like genes plays an important role in the recognition and transport of their specific substrates.

Full text

PDF
7289

Images in this article

7290Image
on p.7290
7290Image
on p.7290

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ames G. F. Bacterial periplasmic transport systems: structure, mechanism, and evolution. Annu Rev Biochem. 1986;55:397–425. doi: 10.1146/annurev.bi.55.070186.002145. [DOI] [PubMed] [Google Scholar]
  2. Bruggemann E. P., Germann U. A., Gottesman M. M., Pastan I. Two different regions of P-glycoprotein [corrected] are photoaffinity-labeled by azidopine. J Biol Chem. 1989 Sep 15;264(26):15483–15488. [PubMed] [Google Scholar]
  3. Buschman E., Gros P. Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdr1 and mdr2 genes. Mol Cell Biol. 1991 Feb;11(2):595–603. doi: 10.1128/mcb.11.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen C. J., Chin J. E., Ueda K., Clark D. P., Pastan I., Gottesman M. M., Roninson I. B. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell. 1986 Nov 7;47(3):381–389. doi: 10.1016/0092-8674(86)90595-7. [DOI] [PubMed] [Google Scholar]
  5. Choi K. H., Chen C. J., Kriegler M., Roninson I. B. An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene. Cell. 1988 May 20;53(4):519–529. doi: 10.1016/0092-8674(88)90568-5. [DOI] [PubMed] [Google Scholar]
  6. Cornwell M. M., Gottesman M. M., Pastan I. H. Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells. J Biol Chem. 1986 Jun 15;261(17):7921–7928. [PubMed] [Google Scholar]
  7. Cornwell M. M., Safa A. R., Felsted R. L., Gottesman M. M., Pastan I. Membrane vesicles from multidrug-resistant human cancer cells contain a specific 150- to 170-kDa protein detected by photoaffinity labeling. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3847–3850. doi: 10.1073/pnas.83.11.3847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cornwell M. M., Tsuruo T., Gottesman M. M., Pastan I. ATP-binding properties of P glycoprotein from multidrug-resistant KB cells. FASEB J. 1987 Jul;1(1):51–54. doi: 10.1096/fasebj.1.1.2886389. [DOI] [PubMed] [Google Scholar]
  9. Devault A., Gros P. Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities. Mol Cell Biol. 1990 Apr;10(4):1652–1663. doi: 10.1128/mcb.10.4.1652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  11. Endicott J. A., Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem. 1989;58:137–171. doi: 10.1146/annurev.bi.58.070189.001033. [DOI] [PubMed] [Google Scholar]
  12. Foote S. J., Kyle D. E., Martin R. K., Oduola A. M., Forsyth K., Kemp D. J., Cowman A. F. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature. 1990 May 17;345(6272):255–258. doi: 10.1038/345255a0. [DOI] [PubMed] [Google Scholar]
  13. Foote S. J., Thompson J. K., Cowman A. F., Kemp D. J. Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum. Cell. 1989 Jun 16;57(6):921–930. doi: 10.1016/0092-8674(89)90330-9. [DOI] [PubMed] [Google Scholar]
  14. Gottesman M. M., Pastan I. The multidrug transporter, a double-edged sword. J Biol Chem. 1988 Sep 5;263(25):12163–12166. [PubMed] [Google Scholar]
  15. Gros P., Ben Neriah Y. B., Croop J. M., Housman D. E. Isolation and expression of a complementary DNA that confers multidrug resistance. Nature. 1986 Oct 23;323(6090):728–731. doi: 10.1038/323728a0. [DOI] [PubMed] [Google Scholar]
  16. Gros P., Croop J., Housman D. Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell. 1986 Nov 7;47(3):371–380. doi: 10.1016/0092-8674(86)90594-5. [DOI] [PubMed] [Google Scholar]
  17. Gros P., Raymond M., Bell J., Housman D. Cloning and characterization of a second member of the mouse mdr gene family. Mol Cell Biol. 1988 Jul;8(7):2770–2778. doi: 10.1128/mcb.8.7.2770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hamada H., Tsuruo T. Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase. J Biol Chem. 1988 Jan 25;263(3):1454–1458. [PubMed] [Google Scholar]
  19. Hsu S. I., Cohen D., Kirschner L. S., Lothstein L., Hartstein M., Horwitz S. B. Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells. Mol Cell Biol. 1990 Jul;10(7):3596–3606. doi: 10.1128/mcb.10.7.3596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hyde S. C., Emsley P., Hartshorn M. J., Mimmack M. M., Gileadi U., Pearce S. R., Gallagher M. P., Gill D. R., Hubbard R. E., Higgins C. F. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature. 1990 Jul 26;346(6282):362–365. doi: 10.1038/346362a0. [DOI] [PubMed] [Google Scholar]
  21. Kuchler K., Sterne R. E., Thorner J. Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells. EMBO J. 1989 Dec 20;8(13):3973–3984. doi: 10.1002/j.1460-2075.1989.tb08580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McGrath J. P., Varshavsky A. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature. 1989 Aug 3;340(6232):400–404. doi: 10.1038/340400a0. [DOI] [PubMed] [Google Scholar]
  23. Monaco J. J., Cho S., Attaya M. Transport protein genes in the murine MHC: possible implications for antigen processing. Science. 1990 Dec 21;250(4988):1723–1726. doi: 10.1126/science.2270487. [DOI] [PubMed] [Google Scholar]
  24. Naito M., Hamada H., Tsuruo T. ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells. J Biol Chem. 1988 Aug 25;263(24):11887–11891. [PubMed] [Google Scholar]
  25. Qian X. D., Beck W. T. Binding of an optically pure photoaffinity analogue of verapamil, LU-49888, to P-glycoprotein from multidrug-resistant human leukemic cell lines. Cancer Res. 1990 Feb 15;50(4):1132–1137. [PubMed] [Google Scholar]
  26. Raviv Y., Pollard H. B., Bruggemann E. P., Pastan I., Gottesman M. M. Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells. J Biol Chem. 1990 Mar 5;265(7):3975–3980. [PubMed] [Google Scholar]
  27. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  28. Safa A. R., Glover C. J., Sewell J. L., Meyers M. B., Biedler J. L., Felsted R. L. Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers. J Biol Chem. 1987 Jun 5;262(16):7884–7888. [PubMed] [Google Scholar]
  29. Safa A. R. Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7187–7191. doi: 10.1073/pnas.85.19.7187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schurr E., Raymond M., Bell J. C., Gros P. Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse mdr1 cDNA. Cancer Res. 1989 May 15;49(10):2729–2733. [PubMed] [Google Scholar]
  31. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  32. Ueda K., Cardarelli C., Gottesman M. M., Pastan I. Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci U S A. 1987 May;84(9):3004–3008. doi: 10.1073/pnas.84.9.3004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yang C. P., Cohen D., Greenberger L. M., Hsu S. I., Horwitz S. B. Differential transport properties of two mdr gene products are distinguished by progesterone. J Biol Chem. 1990 Jun 25;265(18):10282–10288. [PubMed] [Google Scholar]
  34. Yoshimura A., Kuwazuru Y., Sumizawa T., Ichikawa M., Ikeda S., Uda T., Akiyama S. Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies. J Biol Chem. 1989 Sep 25;264(27):16282–16291. [PubMed] [Google Scholar]
  35. van der Bliek A. M., Kooiman P. M., Schneider C., Borst P. Sequence of mdr3 cDNA encoding a human P-glycoprotein. Gene. 1988 Nov 30;71(2):401–411. doi: 10.1016/0378-1119(88)90057-1. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES