Localization of the forskolin labeling sites to both halves of P-glycoprotein: similarity of the sites labeled by forskolin and prazosin.
An iodinated derivative of forskolin, 6-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]forskolin ([125I]6-AIPP-Fsk), photolabels the multidrug efflux pump P-glycoprotein in membranes prepared from the multidrug-resistant cell lines KB-V1 and KB-C1. The labeling site for [125I]6-AIPP-Fsk was localized by immunoprecipitation of tryptic fragments of P-glycoprotein labeled in KB-C1 membranes. A 6-kDa, photolabeled, tryptic fragment was immunoprecipitated by antiserum raised against residues 348-419 of P-glycoprotein, PEPG9, but not by antisera raised against flanking regions PEPG7 and PEPG11. A peptide that corresponds to residues 343-359 of P-glycoprotein inhibited immunoprecipitation of the 6-kDa fragment by antiserum against PEPG9 but had no effect on the immunoprecipitation of photolabeled fragments by antiserum against PEPG7. A second peptide, corresponding to residues 360-376, had no effect on the immunoprecipitation by antiserum against PEPG9. [125I]6-AIPP-Fsk labels the carboxyl-terminal half of P-glycoprotein, because low molecular mass tryptic fragments were immunoprecipitated by three carboxyl-terminal antisera. Therefore, [125I]6-AIPP-Fsk labels both halves of P-glycoprotein, and labeling in the amino-terminal half can be localized to residues 291-359, which span proposed transmembrane regions 5 and 6. KB-V1 membranes photolabeled with [125I]6-AIPP-Fsk and [125I]iodoarylazidoprazosin were digested with either Staphylococcus aureus V8 protease or chymotrypsin and had similar digestion patterns, suggesting that the two drugs label the same sites on P-glycoprotein.
Morris
D I
DI
Molecular Pharmacology Laboratory, Food and Drug Administration, Bethesda, Maryland 20892.
Greenberger
L M
LM
Bruggemann
E P
EP
Cardarelli
C
C
Gottesman
M M
MM
Pastan
I
I
Seamon
K B
KB
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011224:Prazosin / Q000031:analogs & derivatives / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D014357:Trypsin; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D007106:Immune Sera; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D005576:Colforsin; D014357:Trypsin; D011224:Prazosin