Localization of the Iodomycin Binding Site in Hamster P-glycoprotein

Abstract

P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds. This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin. 125 I]iodomycin had been predominantly incorporated into the fragment 230 -312 of isoform I of hamster P-glycoprotein. According to models based on hydropathy plots, the amino acid sequence 230 -312 forms the distal part of transmembrane segment 4, the second cytoplasmic loop, and the proximal part of transmembrane segment 5 in the Nterminal half of P-glycoprotein. The binding site for iodomycin is recognized with high affinity by vinblastine and cyclosporin A. P-glycoprotein, which belongs to the large family of ABC 1 transporters (1), binds and transports a broad range of structurally unrelated compounds (2). Its overexpression may cause the phenomenon of multidrug resistance (MDR) during cancer chemotherapy, whereby the tumor cells become resistant to a variety of antineoplastic agents due to a reduced intracellular accumulation of drugs (2-4). The MDR phenotype can be overcome by modulators, i.e. substances that are bound by P-glycoprotein and inhibit its drug excluding function (3). The medically important substrates of P-glycoprotein comprise anticancer drugs such as Vinca alkaloids and anthracyclines (2-4) and approved drugs that turned out to be potent modulators such as verapamil (5, 6), calcium antagonists (7), and cyclosporins (8).The major issue of how P-glycoprotein can handle so many substrates has been mainly approached by photoaffinity labeling and mutagenesis studies. Mutants that arose spontaneously during drug selection of cells and thereby changed their resistance profile were detected in the first cytoplasmic loop of human MDR1 protein (Gly 185 3 Val) (9) and in transmembrane segment (TM) 6 of hamster P-glycoprotein (Gly 338 3 Ala/Ala 339 3 Pro) (10). Active mutagenesis identified further motifs and positions in the N-and C-terminal half of P-glycoprotein which influence substrate specificity (11, 12). For example, the exchange of amino acids in the first cytoplasmic loop (11), TM5, TM6, TM10, TM12 (13-16), and the cytosolic linker peptide (17) resulted in all in an altered multidrug resistance phenotype, suggesting that during binding and transport the substrate is recognized by multiple residues located either in the cytosolic, membraneous, or ectoplasmic domains.Hydropathy plots deduced from the primary sequence predict that P-glycoprotein consists principally of two symmetrical halves, each of which contain a membrane domain with six membrane-spanning segments and a subsequent cytosolic nucleotide binding fold (18 -20). The topology of P-glycoprotein in vivo, however, may be more variable than predicted, e.g. under certain experimental conditions, TM segments were detected outside the membrane and loops postulated to face the cytosol were foun...

Flavonoids: A class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein

“…5A-C). (47,48), one of which is located on the cytosolic side of the transporter (49). The partial antagonism observed here between kaempferide and RU 486, and the higher antagonism of kaempferide against MANT-ATP as compared with ATP, for binding to H 6 -NBD2, indicates that the flavonoid indeed binds to the steroid-interacting hydrophobic region.…”

The Packing of the Transmembrane Segments of Human Multidrug Resistance P-glycoprotein Is Revealed by Disulfide Cross-linking Analysis

“…Many mutations in TMs 4, 5, and 6, and in TMs 10, 11, and 12, have been reported to alter the substrate specificity or activity of the transporter (23)(24)(25)(26)(27)(28)(29)(30)(31). Labeling studies with photoactive analogs of P-gp substrates also suggest that TMs 4, 5, and 6 and TMs 10, 11, and 12 may participate in drug-protein interactions (32)(33)(34)(35)(36). The model in Fig.…”

Identification of P-glycoprotein Mutations Causing a Loss of Steroid Recognition and Transport

“…These regions include the transmembrane domains 5 and 6, as well as the 11 and 12 domains. More recently, Demmer et al (52) used iodomycin, a modified form of daunomycin, to map an anthracycline binding site that included the 4 and 5 transmembrane domains of a hamster Pgp. Our results are in accordance with that observation.…”