Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Abstract

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [ 125 I]iodoarylazidoprazosin ([ 125 I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [ 125 I]IAAP binding to the N- and C-terminal halves. cis ( Z )-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [ 125 I]IAAP for the C-terminal half of the protein (C-site) by reducing the K d from 20 to 6 nM without changing the labeling or affinity ( K d = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [ 125 I]IAAP binding to the C-site was increased 5- to 6-fold by cis ( Z )-flupentixol without any effect on the N-site. In addition, [ 125 I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.