Identification of the domains of photoincorporation of the 3'- and 7- benzophenone analogues of taxol in the carboxyl-terminal half of murine mdr1b P-glycoprotein

Qing Wu, Pierre Yves Bounaud, Scott D. Kuduk, Chia-Ping H. Yang, Iwao Ojima, Susan Band Horwitz, George A. Orr

Research output: Contribution to journalArticle

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Abstract

P-glycoprotein is an ATP-dependent drug-efflux pump that can transport a diverse range of structurally and functionally unrelated hydrophobic 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 between the murine mdr1b P-glycoprotein and two photoreactive Taxol analogues have been mapped by a combination of CNBr digestion and immunoprecipitation studies. We had demonstrated previously that the 3'-p-benzoyldihydrocinnamoyl (BzDC) analogue of Taxol specifically photolabeled mdr1b P-glycoprotein and now show that the corresponding C-7 analogue likewise specifically photoincorporates into the transporter. CNBr digestion of both photolabeled P-glycoproteins gave rise to an approximate 10 kDa tritium-labeled peptide, each of which was a distinct polypeptide. The CNBr fragment generated from the 3'-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab7) raised against amino acid residues 1008-1019 of the mdr1b isoform. In contrast, 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 reactions was demonstrated by showing that the presence of the appropriate synthetic peptide blocked the immunoprecipitation. Moreover when the antibodies were reversed, no immunoprecipitation occurred. Based on the deduced amino acid sequence of mdr1b P-glycoprotein, and its hydropathy plot analysis, our data indicated that the 3'-BzDC group photoincorporates into amino acid residues 985-1088, a region of the transporter that includes half of TM 12 and terminates just after the Walker A motif in the second nucleotide binding fold. The 7-BzDC group photoincorporates into amino acid residues 683-760, a region of the transporter that includes all of TM 7 and half of TM 8 plus the intervening extracellular loop.

Original languageEnglish (US)
Pages (from-to)11272-11279
Number of pages8
JournalBiochemistry
Volume37
Issue number32
DOIs
StatePublished - Aug 11 1998

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P-Glycoprotein
Paclitaxel
Amino Acids
Immunoprecipitation
Peptides
Antibodies
Digestion
Nucleotides
P-Glycoproteins
Tritium
Cell membranes
oxybenzone
benzophenone
Amino Acid Sequence
Protein Isoforms
Adenosine Triphosphate
Cell Membrane
Pumps
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of the domains of photoincorporation of the 3'- and 7- benzophenone analogues of taxol in the carboxyl-terminal half of murine mdr1b P-glycoprotein. / Wu, Qing; Bounaud, Pierre Yves; Kuduk, Scott D.; Yang, Chia-Ping H.; Ojima, Iwao; Band Horwitz, Susan; Orr, George A.

In: Biochemistry, Vol. 37, No. 32, 11.08.1998, p. 11272-11279.

Research output: Contribution to journalArticle

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abstract = "P-glycoprotein is an ATP-dependent drug-efflux pump that can transport a diverse range of structurally and functionally unrelated hydrophobic 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 between the murine mdr1b P-glycoprotein and two photoreactive Taxol analogues have been mapped by a combination of CNBr digestion and immunoprecipitation studies. We had demonstrated previously that the 3'-p-benzoyldihydrocinnamoyl (BzDC) analogue of Taxol specifically photolabeled mdr1b P-glycoprotein and now show that the corresponding C-7 analogue likewise specifically photoincorporates into the transporter. CNBr digestion of both photolabeled P-glycoproteins gave rise to an approximate 10 kDa tritium-labeled peptide, each of which was a distinct polypeptide. The CNBr fragment generated from the 3'-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab7) raised against amino acid residues 1008-1019 of the mdr1b isoform. In contrast, 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 reactions was demonstrated by showing that the presence of the appropriate synthetic peptide blocked the immunoprecipitation. Moreover when the antibodies were reversed, no immunoprecipitation occurred. Based on the deduced amino acid sequence of mdr1b P-glycoprotein, and its hydropathy plot analysis, our data indicated that the 3'-BzDC group photoincorporates into amino acid residues 985-1088, a region of the transporter that includes half of TM 12 and terminates just after the Walker A motif in the second nucleotide binding fold. The 7-BzDC group photoincorporates into amino acid residues 683-760, a region of the transporter that includes all of TM 7 and half of TM 8 plus the intervening extracellular loop.",
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T1 - Identification of the domains of photoincorporation of the 3'- and 7- benzophenone analogues of taxol in the carboxyl-terminal half of murine mdr1b P-glycoprotein

AU - Wu, Qing

AU - Bounaud, Pierre Yves

AU - Kuduk, Scott D.

AU - Yang, Chia-Ping H.

AU - Ojima, Iwao

AU - Band Horwitz, Susan

AU - Orr, George A.

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