Abstract
P-glycoprotein (Pgp), the ATP-binding cassette (ABC) transporter, confers multidrug resistance to cancer cells by extruding cytotoxic natural product amphipathic drugs using the energy of ATP hydrolysis. Our studies are directed toward understanding the mechanism of action of Pgp and recent work deals with the assessment of interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified Pgp suggest that ADP release is the rate-limiting step in the catalytic cycle and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of Pgp, one in the transport of drug and the other in effecting conformational changes so as to reset the transporter for the next catalytic cycle. Detailed kinetic measurements determined that both nucleotide-binding domains behave symmetrically and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time, causing (in this site) a conformational change that drastically decreases (>30-fold) the affinity of the second site for ATP-binding. Thus, the blocking of ATP-binding to the second site while the first one is in catalytic conformation appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by Pgp, and this may be applicable as well to other ABC transporters linked with the development of multidrug resistance.
Similar content being viewed by others
REFERENCES
al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994). J. Biol. Chem. 269, 8986–8992.
Allen, J. D., Brinkhuis, R. F., van Deemter, L., Wijnholds, J., and Schinkel, A. H. (2000). Cancer Res. 60, 5761–5766.
Allikmets, R., Schriml, L. M., Hutchinson, A., Romano-Spica, V., and Dean, M. (1998). Cancer Res. 58, 5337–5339.
Ambudkar, S. V. (1998). Methods Enzymol. 292, 504–514.
Ambudkar, S. V., Cardarelli, C. O., Pashinsky, I., and Stein, W. D. (1997). J. Biol. Chem. 272, 21160–21166.
Ambudkar, S. V., Dey, S., Hrycyna, C. A., Ramachandra, M., Pastan, I., and Gottesman, M. M. (1999). Annu. Rev. Pharmacol. Toxicol. 39, 361–398.
Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1992). Proc. Natl. Acad. Sci. U.S.A. 89, 8472–8476.
Bakos, E., Evers, R., Szakacs, G., Tusnady, G. E., Welker, E., Szabo, K., de Haas, M., van Deemter, L., Borst, P., Varadi, A., and Sarkadi, B. (1998). J. Biol. Chem. 273, 32167–32175.
Borst, P. (1999). Ann. Oncol. 10, 162–164.
Borst, P., and Schinkel, A. H. (1996). Eur. J. Cancer 32A, 985–990.
Borst, P., and Schinkel, A. H. (1997). Trends Genet. 13, 217–222.
Bruggemann, E. P., Currier, S. J., Gottesman, M. M., and Pastan, I. (1992). J. Biol. Chem. 267, 21020–21026.
Bruggemann, E. P., Germann, U. A., Gottesman, M. M., and Pastan, I. (1989). J. Biol. Chem. 264, 15483–15488.
Chen, C. J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986). Cell 47, 381–389.
Cole, S. P., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant, C. E., Almquist, K. C., Stewart, A. J., Kurz, E. U., Duncan, A. M., and Deeley, R. G. (1992). Science 258, 1650–1654.
Cole, S. P., and Deeley, R. G. (1998). Bioessays 20, 931–940.
Dean, M., and Allikmets, R. (2001). J. Bioenerg. Biomembr. 33(6), 449–526.
Demmer, A., Thole, H., Kubesch, P., Brandt, T., Raida, M., Fislage, R., and Tummler, B. (1997). J. Biol. Chem. 272, 20913–20919.
Dey, S., Ramachandra, M., Pastan, I., Gottesman, M. M., and Ambudkar, S. V. (1997). Proc. Natl. Acad. Sci. U.S.A. 94, 10594–10599.
Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000). Embo. J. 19, 5951–5961.
Doyle, L. A., Yang, W., Abruzzo, L. V., Krogmann, T., Gao, Y., Rishi, A. K., and Ross, D. D. (1998). Proc. Natl. Acad. Sci. U.S.A. 95, 15665–15670.
Endicott, J. A., and Ling, V. (1989). Annu. Rev. Biochem. 58, 137–171.
Ford, J. M., and Hait, W. N. (1990). Pharmacol. Rev. 42, 155–199.
Germann, U. A. (1996). Eur. J. Cancer 32A, 927–944.
Gottesman, M. M., Ambudkar, S. V., Ni, B., Aran, J. M., Sugimoto, Y., Cardarelli, C. O., and Pastan, I. (1994). Cold Spring Harb. Symp. Quant. Biol. 59, 677–683.
Gottesman, M. M., Hrycyna, C. A., Schoenlein, P. V., Germann, U. A., and Pastan, I. (1995). Annu. Rev. Genet. 29, 607–649.
Gottesman, M. M., and Pastan, I. (1993). Annu. Rev. Biochem. 62, 385–427.
Greenberger, L. M. (1993). J. Biol. Chem. 268, 11417–11425.
Hafkemeyer, P., Dey, S., Ambudkar, S. V., Hrycyna, C. A., Pastan, I., and Gottesman, M. M. (1998). Biochemistry 37, 16400–16409.
Higgins, C. F., and Gottesman, M. M. (1992). Trends Biochem. Sci. 17, 18–21.
Hrycyna, C. A., Ramachandra, M., Ambudkar, S.V., Ko, Y. H., Pedersen, P. L., Pastan, I., and Gottesman, M. M. (1998). J. Biol. Chem. 273, 16631–16634.
Hrycyna, C. A., Ramachandra, M., Germann, U. A., Cheng, P.W., Pastan, I., and Gottesman, M. M. (1999). Biochemistry 38, 13887–13899.
Johnson, D. R., Finch, R. A., Lin, Z. P., Zeiss, C. J., and Sartorelli, A. C. (2001). Cancer Res. 61, 1469–1476.
Juliano, R. L., and Ling, V. (1976). Biochim. Biophys. Acta. 455, 152–162.
Kerr, K. M., Sauna, Z. E., and Ambudkar, S. V. (2001). J. Biol. Chem. 276, 8657–8664.
Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000). Nature 407, 711–717.
Leith, C. P., Kopecky, K. J., Chen, I. M., Eijdems, L., Slovak, M. L., McConnell, T. S., Head, D. R., Weick, J., Grever, M. R., Appelbaum, F. R., and Willman, C. L. (1999). Blood 94, 1086–1099.
Lelong, I. H., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1994). Biochemistry 33, 8921–8929.
Litman, T., Brangi, M., Hudson, E., Fetsch, P., Abati, A., Ross, D. D., Miyake, K., Resau, J. H., and Bates S. E. (2000). J. Cell. Sci. 113, 2011–2021.
Liu, C. E., Liu, P. Q., and Ames, G. F. L. (1997). J. Biol. Chem. 272, 21883–21891.
Liu, R., and Sharom, F. J. (1996). Biochemistry 35, 11865–11873.
Loe, D. W., Deeley, R. G., and Cole, S. P. (1996). Eur. J. Cancer 32A, 945–957.
Loo, T. W., and Clarke, D. M. (1995a). J. Biol. Chem. 270, 22957–22961.
Loo, T. W., and Clarke, D. M. (1995b). J. Biol. Chem. 270, 21449–21452.
T. W., and Clarke, D. M. (2000). J. Biol. Chem. 275, 39272–39278.
Mimmack, M. L., Gallagher, M. P., Pearce, S. R., Hyde, S. C., Booth, I. R., and Higgins, C. F. (1989). Proc. Natl. Acad. Sci. U.S.A. 86, 8257–8261.
Miyake, K., Mickley, L., Litman, T., Zhan, Z., Robey, R., Cristensen, B., Brangi, M., Greenberger, L., Dean, M., Fojo, T., and Bates, S. E. (1999). Cancer Res. 59, 8–13.
Morris, D. I., Greenberger, L. M., Bruggemann, E. P., Cardarelli, C., Gottesman, M. M., Pastan, I., and Seamon, K. B. (1994). Mol. Pharmacol. 46, 329–337.
Ng, I. O., Lam, K. Y., Ng, M., Kwong, D. L., and Sham, J. S. (1998). Cancer 83, 851–857.
Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000). Nature 407, 703–710.
Ramachandra, M., Ambudkar, S. V., Chen, D., Hrycyna, C. A., Dey, S., Gottesman, M. M., and Pastan, I. (1998). Biochemistry 37, 5010–5019.
Ramachandra, M., Ambudkar, S. V., Gottesman, M. M., Pastan, I., and Hrycyna, C. A. (1996). Mol. Biol. Cell 7, 1485–1498.
Roepe, P. D. (1995). Biochim. Biophys. Acta 1241, 385–405.
Ruetz, S., and Gros, P. (1994). J. Biol. Chem. 269, 12277–12284.
Sankaran, B., Bhagat, S., and Senior, A. E. (1997). Biochemistry 36, 6847–6853.
Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992). J. Biol. Chem. 267, 4854–4858.
Sauna, Z. E., and Ambudkar, S. V. (2000). Proc. Natl. Acad. Sci. U.S.A. 97, 2515–2520.
Sauna, Z. E., and Ambudkar, S. V. (2001). J. Biol. Chem. 276, 11653–11661.
Sauna, Z. E., Smith, M. M., Muller, M., and Ambudkar, S. V. (2001). J. Biol. Chem. 276, 21199–21208.
Scarborough, G. A. (1995). J. Bioenerg. Biomembr. 27, 37–41.
Schinkel, A. H. (1997). Semin. Cancer Biol. 8, 161–170.
Schinkel, A. H., Mayer, U., Wagenaar, E., Mol, C. A., van Deemter, L., Smit, J. J., van der Valk, M. A., Voordouw, A. C., Spits, H., van Tellingen, O., Zijlmans, J. M., Fibbe, W. E., and Borst, P. (1997). Proc. Natl. Acad. Sci. U.S.A. 94, 4028–4033.
Schinkel, A. H., Smit, J. J., van Tellingen, O., Beijnen, J. H., Wagenaar, E., van Deemter, L., Mol, C. A., van der Valk, M. A., Robanus-Maandag, E. C., te Riele, H. P., Berns, A. J. M., and Borst, P. (1994). Cell 77, 491–502.
Seelig, A. (1998a). Eur. J. Biochem. 251, 252–261.
Seelig, A. (1998b). Int. J. Clin. Pharmacol. Ther. 36, 50–54.
Senior, A. E. (1998). Acta Physiol. Scand. Suppl. 643, 213–218.
Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995a). J. Bioenerg. Biomembr. 27, 31–36.
Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995b). FEBS Lett. 377, 285–289.
Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1998). Methods Enzymol. 292, 514–523.
Senior, A. E., and Gadsby, D. C. (1997). Semin. Cancer Biol. 8, 143–150.
Shapiro, A. B., and Ling, V. (1995). J. Biol. Chem. 270, 16167–16175.
Shapiro, A. B., and Ling, V. (1998). Eur. J. Biochem. 254, 189–193.
Sharom, F. J. (1997). J. Membr. Biol. 160, 161–175.
Sharom, F. J., Liu, R., Romsicki, Y., and Lu, P. (1999). Biochim. Biophys. Acta 1461, 327–345.
Sharom, F. J., Yu, X., Chu, J. W., and Doige, C. A. (1995). Biochem. J. 308, 381–390.
Sharom, F. J., Yu, X., and Doige, C. A. (1993). J. Biol. Chem. 268, 24197–24202.
Smith, C. A., and Rayment, I. (1996). Biochemistry 35, 5404–5417.
Szabo, K., Welker, E., Bakos, Muller, M., Roninson, I., Varadi, A., and Sarkadi, B. (1998). J. Biol. Chem. 273, 10132–10138.
Tan, B., Piwnica-Worms, D., and Ratner, L. (2000). Curr. Opin. Oncol. 12, 450–458.
Trock, B. J., Leonessa, F., and Clarke, R. (1997). J. Natl. Cancer Inst. 89, 917–931.
Tsuruo, T., Iida, H., Tsukagoshi, S., and Sakurai, Y. (1982). Cancer Res. 42, 4730–4733.
Tusnady, G. E., Bakos, E., Varadi, A., and Sarkadi, B. (1997). FEBS Lett. 402, 1–3.
Urbatsch, I. L., al-Shawi, M. K., and Senior, A. E. (1994). Biochemistry 33, 7069–7076.
Urbatsch, I. L., Beaudet, L., Carrier, I., and Gros, P. (1998). Biochemistry 37, 4592–4602.
Urbatsch, I. L., Gimi, K., Wilke-Mounts, S., and Senior, A. E. (2000). J. Biol. Chem. 275, 25031–25038.
Urbatsch, I. L., Sankaran, B., Bhagat, S., and Senior, A. E. (1995a). J. Biol. Chem. 270, 26956–26961.
Urbatsch, I. L., Sankaran, B., Weber, J., and Senior, A. E. (1995b). J. Biol. Chem. 270, 19383–19390.
van der Zee, A. G., Hollema, H., Suurmeijer, A. J., Krans, M., Sluiter, W. J., Willemse, P. H., Aalders, J. G., and de Vries, E. G. (1995). J. Clin. Oncol. 13, 70–78.
Yuen, A. R., and Sikic, B. I. (1994). J. Clin. Oncol. 12, 2453–2459.
Zhou, T., Radaev, S., Rosen, B. P., and Gatti, D. L. (2000). Embo. J. 19, 4838–4845.
Zochbauer, S., Gsur, A., Brunner, R., Kyrle, P. A., Lechner, K., and Pirker, R. (1994). Leukemia 8, 974–977.
Rights and permissions
About this article
Cite this article
Sauna, Z.E., Smith, M.M., Müller, M. et al. The Mechanism of Action of Multidrug-Resistance-Linked P-Glycoprotein. J Bioenerg Biomembr 33, 481–491 (2001). https://doi.org/10.1023/A:1012875105006
Issue Date:
DOI: https://doi.org/10.1023/A:1012875105006