Role of the NH₂-terminal Membrane Spanning Domain of Multidrug Resistance Protein 1/ABCC1 in Protein Processing and Trafficking

Abstract

Multidrug resistance protein (MRP)1/ABCC1 transports organic anionic conjugates and confers resistance to cytotoxic xenobiotics. In addition to two membrane spanning domains (MSDs) typical of most ATP-binding cassette (ABC) transporters, MRP1 has a third MSD (MSD0) of unknown function. Unlike some topologically similar ABCC proteins, removal of MSD0 has minimal effect on function, nor does it prevent MRP1 from trafficking to basolateral membranes in polarized cells. However, we find that independent of cell type, the truncated protein accumulates in early/recycling endosomes. Using a real-time internalization assay, we demonstrate that MSD0 is important for MRP1 retention in, or recycling to, the plasma membrane. We also show that MSD0 traffics independently to the cell surface and promotes membrane localization of the core-region of MRP1 when the two protein fragments are coexpressed. Finally, we demonstrate that MSD0 becomes essential for trafficking of MRP1 when the COOH-terminal region of the protein is mutated. These studies demonstrate that MSD0 and the COOH-terminal region contain redundant trafficking signals, which only become essential when one or the other region is missing or is mutated. These data explain apparent differences in the trafficking requirement for MSD0 and the COOH-terminal region of MRP1 compared with other ABCC proteins. INTRODUCTIONMultidrug resistance protein (MRP)1/ABCC1 is a member of the "C" branch of the ATP-binding cassette (ABC) superfamily. When overexpressed in various cell types, MRP1 confers resistance to structurally diverse natural product cytotoxins, as well a certain heavy metal oxyanions and antimetabolites . The protein also has been detected in tumors derived from many different cell types and may be an important factor in the failure of chemotherapy in treating some forms of cancer (Bates, 2003). Many potential physiological and exogenous substrates of MRP1 are organic anion conjugates with glutathione (GSH), glucuronate, or sulfate . In addition, the protein is capable of ATP-dependent, GSH-stimulated transport of various unconjugated hydrophobic substrates (Loe et al., , 1997Renes et al., 1999), as well as certain glucuronate and sulfate conjugates (Sakamoto et al., 1999;Leslie et al., 2001;Qian et al., 2001b).ABC proteins typically consist of two tandemly arranged polytopic membrane spanning domains (MSDs) and two cytoplasmic nucleotide binding domains (NBDs) (Higgins, 2001). In addition to the four "core" domains, MRP1 and certain other ABCC proteins contain a third, NH 2 -terminal MSD (MSD0) . Human ABCC proteins with comparable NH 2 -terminal MSDs include the MRP1 homologues MRP2/ABCC2, MRP3/ABCC3, MRP6/ ABCC6, and MRP7/ABCC10, as well as the sulfonylurea receptors SUR1A/ABCC8 and SUR2A,B/ABCC9 (Tusnady et al., 1997;Haimeur et al., 2004). Despite relatively low sequence similarity, experimental evidence and predictive hydrophobicity determinations suggest that in general, the NH 2 -terminal extensions of these proteins contain five transmembranes (TMs) and ha...

The apical conjugate efflux pump ABCC2 (MRP2)

“…Whereas one report proposed that the PDZ-binding motif is the apical targeting signal of ABCC2 [46], this was not confirmed by all other studies [34, 93,124,183]. These latter studies indicate that the apical targeting motif of ABCC2 is not a distinct, narrowly defined amino acid sequence, but rather appears to be composed of several motifs within different parts of ABCC2 that come together only in the intact protein.…”

“…However, ABCC2, together with ABCC1, ABCC3, ABCC6, and ABCC10, is unique in having an additional amino-terminal polytopic membrane-spanning domain (Fig. 2) that may be required for proper function and localization [34,39,183]. The extracellular location of the amino terminus was first predicted for rat Abcc2 by computational analysis [13] and subsequently proven experimentally [24].…”

Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1)

“…Recently, a detailed study investigating the influence of TMD0 on the subcellular localization of MRP1 revealed that, although TMD0 is not essential for basolateral trafficking of MRP1, deletion of the TMD0 region affects the subcellular distribution of the protein [169]. About 50% of truncated protein was found in the recycling endosomes, whereas this was only 20% in the case of the intact MRP1.…”

“…This finding suggests that TMD0 domain is important for the retention or the recycling of MRP1 to the plasma membrane. It has also been shown that both TMD0 and the remainder of the protein contain routing signals, and TMD0 becomes essential in trafficking when the COOH-terminal region of MRP1 is mutated or truncated [169]. It is important to note that the TMD0 domains in other MRP1-related proteins, including the human MRP2 and yeast cadmium factor-1 (Ycf1), are required for correct apical targeting and for proper vacuolar localization, respectively [37,98].…”

How to overcome ATP-binding cassette drug efflux transporter-mediated drug resistance?

“…It was previously thought that TMD0 had no role in trafficking to the plasma membrane or efflux function, but this view has been revisited by several mechanistic and cell biological reports [52] . It has been shown that TMD0 is important for the processing and trafficking of human MRP1 [53] . Additionally, by mutation studies, Yang et al [54] reported that the amino terminus is required for proper MRP1 function and structure.…”