Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Alice Rothnie, Richard Callaghan, Roger G. Deeley, Susan P.C. Cole

Research output: Contribution to journalArticle

Abstract

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.
Original languageEnglish
Pages (from-to)13906-13914
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number20
Early online date24 Mar 2006
DOIs
Publication statusPublished - 19 May 2006

Keywords

  • binding sites
  • Biological transport
  • tumor cell line
  • estrone
  • glutathione
  • biological models
  • hydrolysis
  • kinetics
  • vincristine
  • multidrug resistance-associated proteins
  • neoplasm metastasis
  • protein binding

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