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Biochemistry. 1997 Dec 9;36(49):15208-15.

Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site.

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1
Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, UPR 412 du Centre National de la Recherche Scientifique, Lyon, France.

Abstract

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.

PMID:
9398248
DOI:
10.1021/bi9718696
[Indexed for MEDLINE]
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