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

Lee M. Greenberger, Chia-Ping H. Yang, Esther Gindin, Susan Band Horwitz

Research output: Contribution to journalArticle

93 Citations (Scopus)

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 photo-affinity analog for P-glycoprotein, 2-[4-(4-azido-3-[ 125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline ([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 - Mar 15 1990

Fingerprint

P-Glycoprotein
Calcium Channels
Adrenergic Receptors
Antineoplastic Agents
Digestion
Vinblastine
Prazosin
Calcium Channel Blockers
Chymotrypsin
Verapamil
Peptides
Colchicine
Multiple Drug Resistance
Dactinomycin
Drug Resistance
Pharmaceutical Preparations
Doxorubicin
Membrane Proteins
Binding Sites
Pumps

ASJC Scopus subject areas

  • Biochemistry

Cite this

Photoaffinity probes for the α1-adrenergic receptor and the calcium channel bind to a common domain in P-glycoprotein. / Greenberger, Lee M.; Yang, Chia-Ping H.; Gindin, Esther; Band Horwitz, Susan.

In: Journal of Biological Chemistry, Vol. 265, No. 8, 15.03.1990, p. 4394-4401.

Research output: Contribution to journalArticle

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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 photo-affinity analog for P-glycoprotein, 2-[4-(4-azido-3-[ 125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline ([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.",
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AU - Band Horwitz, Susan

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