Role of the N-terminal Transmembrane Region of the Multidrug Resistance Protein MRP2 in Routing to the Apical Membrane in MDCKII Cells

Abstract

In polarized cells, the multidrug resistance protein MRP2 is localized in the apical plasma membrane, whereas MRP1, another multidrug resistance protein (MRP) family member, is localized in the basolateral membrane. MRP1 and MRP2 are thought to contain an N-terminal region of five transmembrane segments (TMD 0 ) coupled to 2 times six transmembrane segments via an intracellular loop (L 0 ). We previously demonstrated for MRP1 that a mutant lacking TMD 0 but still containing L 0 , called L 0 ⌬MRP1, was functional and routed to the lateral plasma membrane. To investigate the role of the TMD 0 L 0 region of MRP2 in routing to the apical membrane, we generated mutants similar to those made for MRP1. In contrast to L 0 ⌬MRP1, L 0 ⌬MRP2 was associated with an intracellular compartment, most likely endosomes. Co-expression with TMD 0 , however, resulted in apical localization of L 0 ⌬MRP2 and transport activity. Uptake experiments with vesicles containing L 0 ⌬MRP2 demonstrated that the molecule is able to transport LTC 4 . An MRP2 mutant without TMD 0 L 0 , ⌬MRP2, was only core-glycosylated and localized intracellularly. Co-expression of ⌬MRP2 with TMD 0 L 0 resulted in an increased protein level of ⌬MRP2, full glycosylation of the protein, routing to the apical membrane, and transport activity. Our results suggest that the TMD 0 region is required for routing to or stable association with the apical membrane.Several members of the ATP-binding cassette superfamily of transporter proteins are able to confer multidrug resistance to tumor cells. Examples are MDR1 P-glycoprotein (1) and members of the multidrug resistance protein (MRP) 1 family, MRP1and MRP2 (2, 3). Whereas MDR1 Pgp preferably transports large amphipathic molecules (4), MRP1 and MRP2 transport in addition acidic compounds with a large hydrophobic moiety such as drugs conjugated with glutathione, glucuronide, or sulfate (5-7). MRP1-and MRP2-mediated transport of cytotoxic drugs not known to be conjugated to negatively charged ligands is most likely due to co-transport with reduced glutathione (8 -12). Besides MRP1 and MRP2, there are seven other MRP homologs expressed in humans, designated MRP3 to -9 (13-15). MRP3 to -5 can confer resistance against some anticancer drugs and are able to transport certain organic anions (16 -21).The predicted membrane topology of MDR1 Pgp and MRPs is different. Whereas MDR1 Pgp and MRPs share a similar core region consisting of two times six transmembrane regions and two intracellular ATP-binding cassettes, MRP1 and MRP2, and several other members of the MRP family contain an extra amino-terminal domain of about 280 amino acids (22-26) (see also Fig. 1). This latter domain is thought to consist of five transmembrane segments and is bound to the core region via an intracellular loop (L 0 ) of ϳ80 amino acids. By producing various mutants of MRP1 in baculovirus-infected insect cells, we and others previously showed that the MRP1 core, called ⌬MRP1, was inactive. Co-expression of the core (⌬MRP1) with TMD 0 L 0 , however, resu...

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

“…Although the mechanism of MRP1 turnover at the plasma membrane is not known, defects in recycling of the wild-type protein have been observed in cisplatin-selected cell lines (Liang et al, 2003). The accumulation of MRP1, MRP2, and the yeast Ycf1 in endosomes in the absence of MSD0, coupled with the observation that appropriate membrane localization of NH 2 -terminal truncations of these proteins can be restored by coexpression with their respective MSD0, is consistent with a conserved function for this domain in membrane targeting ( Figure 5, C-E) (Fernandez et al, 2002;Mason and Michaelis, 2002). When expressed alone, a hemagglutinin-tagged MRP2 MSD0 fragment could not be detected by confocal microscopy (Fernandez et al, 2002).…”

“…Furthermore, conserved tryptophans in MSD0 have been mutated singly, without affecting the transport activity of the protein (Koike et al, 2002). Nevertheless, studies of three ABCC proteins, SUR1A, MRP2, and Ycf1, indicate that MSD0 has at least a partially conserved role in protein trafficking (Fernandez et al, 2002;Mason and Michaelis, 2002;Babenko and Bryan, 2003). Thus, MRP1, which retains activity and the ability to traffic to the plasma membrane in the absence of MSD0, seems to be the exception (Bakos et al, 1998;Westlake et al, 2003).…”

“…Accumulate in the ER, Golgi, or Lysosomes ABCC mutant proteins with processing defects have been found to be incompletely N-glycosylated as a result of impaired processing through the Golgi (Sharma et al, 1999;Gentzsch and Riordan, 2001;Fernandez et al, 2002;Gentzsch et al, 2002). Consequently, we used PNGaseF treatment and SDS-PAGE to compare the glycosylation status of MRP1 204 -1531 with wild-type MRP1 and a more extensively truncated protein, MRP1 , that we showed previously is retained in the ER (Westlake et al, 2003).…”

“…Presently, a functional role for the NH 2 -terminal MSD has been best characterized in stud- ies of SUR1, where an interaction between MSD0 and the potassium channel, Kir6.2 is required for K ATP -channel plasma membrane trafficking and gating (Otonkoski et al, 1999;Babenko and Bryan, 2003;Chan et al, 2003). In addition, the NH 2 -terminal MSDs of MRP2 and the yeast MRP1 orthologue, Ycf1, are necessary for apical membrane and vacuolar localization, respectively (Fernandez et al, 2002;Mason and Michaelis, 2002).…”

ABCC8 and ABCC9: ABC transporters that regulate K+ channels

“…In some ABCC proteins TMD0 is required for correct trafficking [53,91], whereas in MRP1, L0, but not TMD0, is required for transport activity [15] and is part of the glutathione binding site [75]. Coexpression of SUR1 TMD0 with poorly active (K IR 6.2) 4 pores demonstrated partial restoration of function, including an increase in their maximum channel open probability (P Omax ) and the restoration of bursting activity; immunoprecipitation experiments confirmed TMD0 forms complexes with K IR 6.2 [7,31].…”