IDENTIFICATION OF THE DOMAINS OF PHOTOINCORPORATION OF THE 3'-BENZOPHENONE AND 7-BENZOPHENONE ANALOGS OF TAXOL IN THE CARBOXYL-TERMINAL HALF OF MURINE MDR1B P-GLYCOPROTEIN

P-glycoprotein is an ATP-dependent drug-efflux pump that can transport a diverse range of structurally and functionally unrelated hydrophobi c compounds across the plasma membrane. The transporter is composed of two homologous halves, each containing a nucleotide binding fold and six putative transmembrane spanning segments. The contact domains betw een the murine mdr1b P-glycoprotein and two photoreactive Taxol analog ues have been mapped by a combination of CNBr digestion and immunoprec ipitation studies. We had demonstrated previously that the 3'-p-benzoy ldihydrocinnamoyl (BzDC) analogue of Taxol specifically photolabeled m dr1b P-glycoprotein and now show that the corresponding C-7 analogue l ikewise specifically photoincorporates into the transporter. CNBr dige stion of both photolabeled P-glycoproteins gave rise to an approximate 10 kDa tritium-labeled peptide, each of which was a distinct polypept ide. The CNBr fragment generated from the 3'-BzDC-Taxol-photolabeled P -glycoprotein was immunoprecipitated by a polyclonal antibody (Ab7) ra ised against amino acid residues 1008-1019 of the mdr1b isoform. In co ntrast, the CNBr fragment generated from the 7-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab4) raised against amino acid residues 740-750. The specificity of these r eactions was demonstrated by showing that the presence of the appropri ate synthetic peptide blocked the immunoprecipitation, Moreover when t he antibodies were reversed, no immunoprecipitation occurred. Based on the deduced amino acid sequence of mdr1b P-glycoprotein, and its hydr opathy plot analysis, our data indicated that the 3'-BzDC group photoi ncorporates into amino acid residues 985-1088, a region of the transpo rter that includes half of TM 12 and terminates just after the Walker A motif in the second nucleotide binding fold. The 7-BzDC group photoi ncorporates into amino acid residues 683-760, a region of the transpor ter that includes all of TM 7 and half of TM 8 plus the intervening ex tracellular loop.