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, 5]iodophenyl)propionamido]ethyl] carbamyl]forskolin ([I-125]6-AIPP-Fsk), photolabels the multidrug effl ux pump P-glycoprotein in membranes prepared from the multidrug-resist ant cell lines KB-V1 and KB-C1. The labeling site for [I-125]6-AIPP-Fs k was localized by immunoprecipitation of tryptic fragments of P-glyco protein labeled in KB-C1 membranes. A 6-kDa, photolabeled, tryptic fra gment was immunoprecipitated by antiserum raised against residues 348- 419 of P-glycoprotein, PEPG9, but not by antisera raised against flank ing regions PEPG7 and PEPG11. A peptide that corresponds to residues 3 43-359 of P-glycoprotein inhibited immunoprecipitation of the 6-kDa fr agment by antiserum against PEPG9 but had no effect on the immunopreci pitation of photolabeled fragments by antiserum against PEPG7. A secon d peptide, corresponding to residues 360-376, had no effect on the imm unoprecipitation by antiserum against PEPG9. [I-125]6-AIPP-Fsk labels the carboxyl-terminal half of P-glycoprotein, because low molecular ma ss tryptic fragments were immunoprecipitated by three carboxyl-termina l antisera. Therefore, [I-125]6-AlPP-Fsk labels both halves of P-glyco protein, and labeling in the aminoterminal half can be localized to re sidues 291-359, which span proposed transmembrane regions 5 and 6. KB- V1 membranes photolabeled with [I-125]6-AIPP-Fsk and [I-125]iodoarylaz idoprazosin were digested with either Staphlyococcus aureus V8 proteas e or chymotrypsin and had similar digestion patterns, suggesting that the two drugs label the same sites on P-glycoprotein.