TY - JOUR
T1 - Nucleotide-induced conformational changes in the human multidrug resistance protein MRP1 are related to the capacity of chemotherapeutic drugs to accumulate or not in resistant cells
AU - Manciu, Liliana
AU - Chang, Xiu Bao
AU - Riordan, John R.
AU - Buyse, Frédéric
AU - Ruysschaert, Jean Marie
N1 - Funding Information:
L.M. is a recipient of financial support from Fonds pour l’Encouragement de la Recherche Scientifique dans l’Industrie et l’Agriculture (FRIA).
PY - 2001/3/23
Y1 - 2001/3/23
N2 - Intracellular accumulation of anthracycline derivatives was measured in a human embryonic kidney cell line (HEK) and a resistant subline (HEK/multidrug resistance protein (MRP1)) overexpressing MRP1 at the plasma membrane surface. Two compounds (daunorubicin and doxorubicin) were rejected outside the multidrug-resistant cells. On the contrary, three compounds (4′-deoxy-4′-iodo-doxorubicin, 4-demethoxy-daunorubicin and 3′-(3-methoxymorpholino)doxorubicin) accumulated equally within sensitive HEK cells and resistant HEK/MRP1 cells. Our main objective here was to characterize the MRP1 conformational changes mediated by the binding of these anthracycline derivatives and to determine whether these conformational changes are related to MRP1-mediated drug transport. MRP1 was reconstituted in lipid vesicles as previously described [Manciu, L., Chang, X.B., Riordan, J.R. and Ruysschaert, J.-M. (2000) Biochemistry 39, 13026-13033]. The reconstituted protein was shown to conserve its ATPase and drug transport activity. Acrylamide quenching of Trp fluorescence was used to monitor drug-dependent conformational changes. Binding of drugs (4-demethoxy-daunorubicin and 3′-(3-methoxymorpholino)doxorubicin) which accumulate in resistant cells immobilizes MRP1 in a conformational state that is insensitive to ATP binding whereas drugs rejected outside the resistant cells (daunorubicin, doxorubicin) favor a conformational change which may be a required step in the transport process.
AB - Intracellular accumulation of anthracycline derivatives was measured in a human embryonic kidney cell line (HEK) and a resistant subline (HEK/multidrug resistance protein (MRP1)) overexpressing MRP1 at the plasma membrane surface. Two compounds (daunorubicin and doxorubicin) were rejected outside the multidrug-resistant cells. On the contrary, three compounds (4′-deoxy-4′-iodo-doxorubicin, 4-demethoxy-daunorubicin and 3′-(3-methoxymorpholino)doxorubicin) accumulated equally within sensitive HEK cells and resistant HEK/MRP1 cells. Our main objective here was to characterize the MRP1 conformational changes mediated by the binding of these anthracycline derivatives and to determine whether these conformational changes are related to MRP1-mediated drug transport. MRP1 was reconstituted in lipid vesicles as previously described [Manciu, L., Chang, X.B., Riordan, J.R. and Ruysschaert, J.-M. (2000) Biochemistry 39, 13026-13033]. The reconstituted protein was shown to conserve its ATPase and drug transport activity. Acrylamide quenching of Trp fluorescence was used to monitor drug-dependent conformational changes. Binding of drugs (4-demethoxy-daunorubicin and 3′-(3-methoxymorpholino)doxorubicin) which accumulate in resistant cells immobilizes MRP1 in a conformational state that is insensitive to ATP binding whereas drugs rejected outside the resistant cells (daunorubicin, doxorubicin) favor a conformational change which may be a required step in the transport process.
KW - Anthracycline
KW - Flow cytometry
KW - Multidrug resistance
KW - Multidrug resistance protein 1
KW - Quenching of tryptophan fluorescence
UR - http://www.scopus.com/inward/record.url?scp=0035937483&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035937483&partnerID=8YFLogxK
U2 - 10.1016/S0014-5793(01)02270-0
DO - 10.1016/S0014-5793(01)02270-0
M3 - Article
C2 - 11278000
AN - SCOPUS:0035937483
SN - 0014-5793
VL - 493
SP - 31
EP - 35
JO - FEBS Letters
JF - FEBS Letters
IS - 1
ER -