Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1)

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• The mechanisms of multidrug resistance-associated protein 1 (MRP1) drug binding and transport remain unclear.
• MRP1 plays a crucial role in the efflux of various substrates, contributing to multidrug resistance in cancer therapy.

Purpose of the Study:

• To characterize the interaction between MRP1 and rhodamine 123 (Rh123) using a photoreactive analogue.
• To identify specific binding sites of MRP1-drug interactions.
• To investigate the functional consequences of MRP1-drug binding on cellular drug resistance.

Main Methods:

• Photoaffinity labeling of plasma membranes from MRP1-transfected HeLa cells with [(125)I]iodoaryl azido-rhodamine 123 (IAARh123).
• Immunoprecipitation using MRP1-specific monoclonal antibodies to confirm protein identity.
• Inhibition studies with known MRP1 substrates and inhibitors (Leukotriene C4, MK571, doxorubicin, colchicine, chloroquine).
• Cell growth assays to determine drug resistance (IC50 values).
• Proteolytic digestion and HPLC analysis to map photolabeled peptides.

Main Results:

• Photoaffinity labeling identified a 190 kDa polypeptide as MRP1 in HeLa-MRP1 cells.
• IAARh123 photolabeling of MRP1 was saturable and inhibited by excess IAARh123, Leukotriene C4, and MK571.
• HeLa-MRP1 cells exhibited increased resistance to Rh123 and Etoposide VP16 compared to control HeLa cells.
• Reduced drug accumulation in the cytosol of HeLa-MRP1 cells contributed to cross-resistance to Rh123.
• Proteolytic mapping revealed three radiolabeled peptides, indicating IAARh123 binds to sites in the N- and C-domains of MRP1.

Conclusions:

• IAARh123 directly binds to MRP1 at three distinct sites located in the N- and C-terminal domains.
• IAARh123 serves as a sensitive and specific probe for studying MRP1-drug interactions.
• Understanding these binding sites can inform strategies to overcome MRP1-mediated drug resistance.