Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P-glycoprotein.
P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P-glycoprotein-containing plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125I-labeled peptides were separated by tricine/PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248-312 [transmembrane segment (TM)4 to TM5], 758-800 (beyond TM7 to beyond TM8) and 1160-1218 (after the Walker A motif of the second nucleotide-binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.
Isenberg
B
B
Klinische Forschergruppe, Zentrum Biochemie and Zentrum Kinderheilkunde, Medizinische Hochschule Hannover, Germany.
Thole
H
H
Tümmler
B
B
Demmer
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D018432:Drug Resistance, Multiple; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019920:Photoaffinity Labels; D010777:Photochemistry; D011224:Prazosin / Q000031:analogs & derivatives* / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D001386:Azides; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D019920:Photoaffinity Labels; C041552:azidoprazosin; D011224:Prazosin