Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site.
We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.
Dayan
G
G
Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, UPR 412 du Centre National de la Recherche Scientifique, Lyon, France.
Jault
J M
JM
Baubichon-Cortay
H
H
Baggetto
L G
LG
Renoir
J M
JM
Baulieu
E E
EE
Gros
P
P
Di Pietro
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D019290:Megestrol Acetate / Q000378:metabolism*; D051379:Mice; D015735:Mifepristone / Q000378:metabolism*; D011374:Progesterone / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; D015735:Mifepristone; D011374:Progesterone; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D019290:Megestrol Acetate