Rhodamine 123 Binds to Multiple Sites in the Multidrug Resistance Protein (MRP1)

Abstract

The mechanisms of MRP1-drug binding and transport are not clear. In this study, we have characterized the interaction between MRP1 and rhodamine 123 (Rh123) using the photoreactive-iodinated analogue, [(125)I]iodoaryl azido-rhodamine 123 (or IAARh123). Photoaffinity labeling of plasma membranes from HeLa cells transfected with MRP1 cDNA (HeLa-MRP1) with IAARh123 shows the photolabeling of a 190 kDa polypeptide not labeled in HeLa cells transfected with the vector alone. Immunoprecipitation of a 190 kDa photolabeled protein with MRP1-sepcific monoclonal antibodies (QCRL-1, MRPr1, and MRPm6) confirmed the identity of this protein as MRP1. Analysis of MRP1-IAARh123 interactions showed that photolabeling of membranes from HeLa-MRP1 with increasing concentrations of IAARh123 was saturable, and was inhibited with excess of IAARh123. Furthermore, the photoaffinity labeling of MRP1 with IAARh123 was greatly reduced in the presence of excess Leukotreine C(4) or MK571, but to a lesser extent with excess doxorubicin, colchicine or chloroquine. Cell growth assays showed 5-fold and 14-fold increase in the IC(50) of HeLa-MRP1 to Rh123 and the Etoposide VP16 relative to HeLa cells, respectively. Analysis of Rh123 fluorescence in HeLa and HeLa-MRP1 cells with or without ATP suggests that cross-resistance to Rh123 is in part due to reduced drug accumulation in the cytosol of HeLa-MRP1 cells. Mild digestion of purified IAARh123-photolabeled MRP1 with trypsin showed two large polypeptides (approximately 111 and approximately 85 kDa) resulting from cleavage in the linker domain (L1) connecting the multiple-spanning domains MSD0 and MSD1 to MSD2. Exhaustive proteolysis of purified IAARh123-labeled 85 and 111 kDa polypeptides revealed one (6 kDa) and two (approximately 6 plus 4 kDa) photolabeled peptides, respectively. Resolution of total tryptic digest of IAARh123-labeled MRP1 by HPLC showed three radiolabeled peaks consistent with the three Staphylococcus aureus V8 cleaved peptides from the Cleveland maps. Together, the results of this study show direct binding of IAARh123 to three sites that localize to the N- and C-domains of MRP1. Moreover, IAARh123 provides a sensitive and specific probe to study MRP1-drug interactions.

P‐glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging

“…5b), which indicated background transport of the probe. It is possible that this additional efflux may be mediated by the Multidrug Resistance Protein-1 (MRP-1), which has been reported to also transport this dye (31). These observations support the fact that Rho 123 efflux assay is a poorly selective method to assess low levels of P-gp activity and shall be replaced by a more selective method like UIC2 shift assay.…”

Photolabeling of Human and Murine Multidrug Resistance Protein 1 with the High Affinity Inhibitor [125I]LY475776 and Azidophenacyl-[35S]Glutathione

“…Photolabeling studies of MRP1 using azido derivatives of IACI and IAARh123 followed by partial proteolysis have identified two preferential sites of cross-linking involving peptides containing TM helices 10 and 11 or TM 16 and 17 (31)(32)(33). Our previous photolabeling studies of MRP1 have shown that LTC 4 , like IACI and IAARh123, labels sites in MSD2 and MSD3 (27).…”

“…As an alternative approach to identifying regions of the protein directly involved in substrate binding, MRP1 has been photolabeled by its known high affinity physiological substrate LTC 4 as well as by compounds that are less well characterized with respect to binding and transport (10,12,27,(31)(32)(33)(34). Although these compounds compete with LTC 4 for binding to MRP1, their affinities appear to be considerably lower, and they also interact with the distantly related P-glycoprotein.…”

“…Although these compounds compete with LTC 4 for binding to MRP1, their affinities appear to be considerably lower, and they also interact with the distantly related P-glycoprotein. Two of the compounds, 125 I-labeled N-(hydrocinchonidin-8Ј-yl)-4-azido-2-hydroxybenzamide (IACI) and iodoaryl azido rhodamine123 (IAARh123), bind to the protein in a GSHindependent manner and label sites in both the NH 2 -and COOH-proximal halves of the protein (32,33,35) (27). Both iodinated compounds have been shown to cross-link preferentially to proteolytic peptides that contain either TM helices 10 and 11 or helices 16 and 17 (31).…”

JC‐1, a sensitive probe for a simultaneous detection of P‐glycoprotein activity and apoptosis in leukemic cells

“…So far none of them has been described as a substrate specific for P-gp. Rh 123 is a P-gp substrate, but its also a MRP1 and BCRP substrate (18,19), and Ca-AM is a P-gp and MRP substrate (12). Until now, JC-1 and Syto 16 haven't been described as either a MRP or a BCRP substrate.…”