Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Feb;11(2):595–603. doi: 10.1128/mcb.11.2.595

Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdr1 and mdr2 genes.

E Buschman 1, P Gros 1
PMCID: PMC359710  PMID: 1990275

Abstract

A full-length cDNA clone for the mouse mdr1 gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdr1 which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdr1 and analyzed these chimeras for their capacity to transfer drug resistance. The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdr1. Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdr1 could also confer drug resistance. However, the replacement of either the amino- or carboxy-terminus transmembrane (TM) domain regions of mdr1 by the homologous segments of mdr2 resulted in inactive chimeras. The replacement of as few as two TM domains from either the amino (TM5-6) or the carboxy (TM7-8) half of mdr1 by the homologous mdr2 regions was sufficient to destroy the activity of mdr1. These results suggest that the functional differences detected between mdr1 and mdr2 in our transfection assay reside within the predicted TM domains.

Full text

PDF
595

Images in this article

598Image
on p.598
599Image
on p.599
600Image
on p.600

Selected References

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

  1. Arceci R. J., Baas F., Raponi R., Horwitz S. B., Housman D., Croop J. M. Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus. Mol Reprod Dev. 1990 Feb;25(2):101–109. doi: 10.1002/mrd.1080250202. [DOI] [PubMed] [Google Scholar]
  2. Arceci R. J., Croop J. M., Horwitz S. B., Housman D. The gene encoding multidrug resistance is induced and expressed at high levels during pregnancy in the secretory epithelium of the uterus. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4350–4354. doi: 10.1073/pnas.85.12.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Azzaria M., Schurr E., Gros P. Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance. Mol Cell Biol. 1989 Dec;9(12):5289–5297. doi: 10.1128/mcb.9.12.5289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blackshear P. J., Nairn A. C., Kuo J. F. Protein kinases 1988: a current perspective. FASEB J. 1988 Nov;2(14):2957–2969. doi: 10.1096/fasebj.2.14.2972578. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Chambers T. C., McAvoy E. M., Jacobs J. W., Eilon G. Protein kinase C phosphorylates P-glycoprotein in multidrug resistant human KB carcinoma cells. J Biol Chem. 1990 May 5;265(13):7679–7686. [PubMed] [Google Scholar]
  7. 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]
  8. Chin J. E., Soffir R., Noonan K. E., Choi K., Roninson I. B. Structure and expression of the human MDR (P-glycoprotein) gene family. Mol Cell Biol. 1989 Sep;9(9):3808–3820. doi: 10.1128/mcb.9.9.3808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Croop J. M., Raymond M., Haber D., Devault A., Arceci R. J., Gros P., Housman D. E. The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues. Mol Cell Biol. 1989 Mar;9(3):1346–1350. doi: 10.1128/mcb.9.3.1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dano K. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochim Biophys Acta. 1973 Oct 25;323(3):466–483. doi: 10.1016/0005-2736(73)90191-0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  16. Felmlee T., Pellett S., Welch R. A. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 1985 Jul;163(1):94–105. doi: 10.1128/jb.163.1.94-105.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fojo A., Akiyama S., Gottesman M. M., Pastan I. Reduced drug accumulation in multiply drug-resistant human KB carcinoma cell lines. Cancer Res. 1985 Jul;45(7):3002–3007. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Glaser P., Sakamoto H., Bellalou J., Ullmann A., Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 1988 Dec 1;7(12):3997–4004. doi: 10.1002/j.1460-2075.1988.tb03288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goldstein L. J., Galski H., Fojo A., Willingham M., Lai S. L., Gazdar A., Pirker R., Green A., Crist W., Brodeur G. M. Expression of a multidrug resistance gene in human cancers. J Natl Cancer Inst. 1989 Jan 18;81(2):116–124. doi: 10.1093/jnci/81.2.116. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. Horio M., Chin K. V., Currier S. J., Goldenberg S., Williams C., Pastan I., Gottesman M. M., Handler J. Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia. J Biol Chem. 1989 Sep 5;264(25):14880–14884. [PubMed] [Google Scholar]
  28. Horio M., Gottesman M. M., Pastan I. ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc Natl Acad Sci U S A. 1988 May;85(10):3580–3584. doi: 10.1073/pnas.85.10.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Hsu S. I., Lothstein L., Horwitz S. B. Differential overexpression of three mdr gene family members in multidrug-resistant J774.2 mouse cells. Evidence that distinct P-glycoprotein precursors are encoded by unique mdr genes. J Biol Chem. 1989 Jul 15;264(20):12053–12062. [PubMed] [Google Scholar]
  31. 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]
  32. Kamimoto Y., Gatmaitan Z., Hsu J., Arias I. M. The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles. J Biol Chem. 1989 Jul 15;264(20):11693–11698. [PubMed] [Google Scholar]
  33. 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]
  34. Lemontt J. F., Azzaria M., Gros P. Increased mdr gene expression and decreased drug accumulation in multidrug-resistant human melanoma cells. Cancer Res. 1988 Nov 15;48(22):6348–6353. [PubMed] [Google Scholar]
  35. Ling V., Thompson L. H. Reduced permeability in CHO cells as a mechanism of resistance to colchicine. J Cell Physiol. 1974 Feb;83(1):103–116. doi: 10.1002/jcp.1040830114. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Mellado W., Horwitz S. B. Phosphorylation of the multidrug resistance associated glycoprotein. Biochemistry. 1987 Nov 3;26(22):6900–6904. doi: 10.1021/bi00396a005. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Naito M., Yusa K., Tsuruo T. Steroid hormones inhibit binding of Vinca alkaloid to multidrug resistance related P-glycoprotein. Biochem Biophys Res Commun. 1989 Feb 15;158(3):1066–1071. doi: 10.1016/0006-291x(89)92830-1. [DOI] [PubMed] [Google Scholar]
  40. Ng W. F., Sarangi F., Zastawny R. L., Veinot-Drebot L., Ling V. Identification of members of the P-glycoprotein multigene family. Mol Cell Biol. 1989 Mar;9(3):1224–1232. doi: 10.1128/mcb.9.3.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pollard J. W., Stanners C. P. Characterization of cell lines showing growth control isolated from both the wild type and a leucyl-tRNA synthetase mutant of Chinese hamster ovary cells. J Cell Physiol. 1979 Mar;98(3):571–585. doi: 10.1002/jcp.1040980315. [DOI] [PubMed] [Google Scholar]
  42. Raymond M., Rose E., Housman D. E., Gros P. Physical mapping, amplification, and overexpression of the mouse mdr gene family in multidrug-resistant cells. Mol Cell Biol. 1990 Apr;10(4):1642–1651. doi: 10.1128/mcb.10.4.1642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Riordan J. R., Deuchars K., Kartner N., Alon N., Trent J., Ling V. Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines. 1985 Aug 29-Sep 4Nature. 316(6031):817–819. doi: 10.1038/316817a0. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Roninson I. B., Chin J. E., Choi K. G., Gros P., Housman D. E., Fojo A., Shen D. W., Gottesman M. M., Pastan I. Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4538–4542. doi: 10.1073/pnas.83.12.4538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Safa A. R., Glover C. J., Meyers M. B., Biedler J. L., Felsted R. L. Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells. J Biol Chem. 1986 May 15;261(14):6137–6140. [PubMed] [Google Scholar]
  47. Safa A. R., Mehta N. D., Agresti M. Photoaffinity labeling of P-glycoprotein in multidrug resistant cells with photoactive analogs of colchicine. Biochem Biophys Res Commun. 1989 Aug 15;162(3):1402–1408. doi: 10.1016/0006-291x(89)90830-9. [DOI] [PubMed] [Google Scholar]
  48. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. Strathdee C. A., Lo R. Y. Cloning, nucleotide sequence, and characterization of genes encoding the secretion function of the Pasteurella haemolytica leukotoxin determinant. J Bacteriol. 1989 Feb;171(2):916–928. doi: 10.1128/jb.171.2.916-928.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wong G. G., Witek J. S., Temple P. A., Wilkens K. M., Leary A. C., Luxenberg D. P., Jones S. S., Brown E. L., Kay R. M., Orr E. C. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science. 1985 May 17;228(4701):810–815. doi: 10.1126/science.3923623. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. 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 Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES