Photoaffinity probes for the α1-adrenergic receptor and the calcium channel bind to a common domain in P-glycoprotein

L. M. Greenberger, C. P.H. Yang, E. Gindin, S. Band Horwitz

Research output: Contribution to journalArticle

94 Scopus citations

Abstract

P-glycoprotein is a 130-180-kDa integral membrane protein that is overproduced in multidrug-resistant cells. The protein appears to act as an energy-dependent drug efflux pump that has broad specificity for structurally diverse hydrophobic antitumor drugs. Many agents, such as the calcium channel blocker verapamil, reverse multidrug resistance and also interact with P-glycoprotein. The goal of this work was to determine if a common binding site participates in the transport of antitumor drugs and/or the reversal of drug resistance. This was done by comparing the peptide maps of P-glycoprotein (encoded by mdr1b) after it was labeled with a photoactive calcium channel blocker, [3H]azidopine, and a newly identified photoaffinity analog for P-glycoprotein, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7-dimet hoxyquinazoline ([125I]iodoaryl azidoprazosin). [125I]Iodoaryl azidoprazosin, which classically has been used to identify the α1-adrenergic receptor, bound to P-glycoprotein and was preferentially competed by vinblastine > actinomycin D > doxorubicin > colchicine. Peptide maps derived from P-glycoprotein labeled with [3H]azidopine or [125I]iodoaryl azidoprazosin were identical. After maximal digestion under conditions for Cleveland mapping, a single major 6-kDa fragment was obtained after digestion with V8 protease, whereas two major fragments, 6.5 and 5.5 kDa, were detected after digestion with chymotrypsin. The 6.0-kDa V8 fragment and the 6.5-kDa chymotrypsin fragment were both found when P-glycoprotein encoded by mdr1a and mdr1b was compared. Despite its specific interaction with P-glycoprotein, neither iodoaryl azidoprazosin nor prazosin markedly reversed resistance compared with verapamil or azidopine. Further, multidrug-resistant cells were 900-fold resistant to vinblastine but only 5-fold resistant to prazosin. These data demonstrate that structurally diverse reversal and/or antitumor agents are likely to have differential affinity for a small common domain of P-glycoprotein.

Original languageEnglish (US)
Pages (from-to)4394-4401
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number8
StatePublished - 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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