Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein.
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 [125I]iodoarylazidoprazosin ([125I]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 [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 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 [125I]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, [125I]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.
Dey
S
S
Laboratory of Cell Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Ramachandra
M
M
Pastan
I
I
Gottesman
M M
MM
Ambudkar
S V
SV
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D005475:Flupenthixol / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D007457:Iodine Radioisotopes / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D011224:Prazosin / Q000031:analogs & derivatives* / Q000378:metabolism; D011869:Radioligand Assay; D014638:Vanadates / Q000378:metabolism; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D014638:Vanadates; D014747:Vinblastine; D016572:Cyclosporine; C041552:azidoprazosin; D005475:Flupenthixol; D011224:Prazosin