Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding

ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp 1000) selected with doxorubicin and verapamil. Later studies demonstrated the presence of a point mutation (Arg 4 8 2 to Thr) in ABCG2 in MCF-7/ AdrVp 1000 cells. This mutation was shown to modulate the transport of Rh 123 (rhodamine 123). In the present study, we have used a previously characterized photoreactive drug analogue of Rh 123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg 4 8 2 → Thr mutation on Rh123 binding and transport by ABCG2. Our results show that both wild-type (ABCG2 R 4 8 2 ) and mutant (ABCG2 T 4 8 2 ) ABCG2 bound directly to IAARh123. Surprisingly, however, wild-type ABCG2 R 4 8 2 , which does not transport Rh 123, was more intensely photolabelled than mutant ABCG2 T 4 8 2 . In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2. For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2. Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.