Identification of the Structural and Functional Boundaries of the Multidrug Resistance Protein 1 Cytoplasmic Loop 3

Abstract

Multidrug resistance protein (MRP) 1 is a member of the ABCC branch of the ATP binding cassette (ABC) transporter superfamily that can confer resistance to natural product chemotherapeutic drugs and transport a variety of conjugated organic anions, as well as some unconjugated compounds in a glutathione- (GSH-) dependent manner. In addition to the two tandemly repeated polytopic membrane-spanning domains (MSDs) typical of ABC transporters, MRP1 and its homologues MRP2, -3, -6, and -7 contain a third NH(2)-terminal MSD. The cytoplasmic loop (CL3) connecting this MSD, but apparently not the MSD itself, is required for MRP1 leukotriene C(4) (LTC(4)) transport activity, substrate binding and appropriate trafficking of the protein to the basolateral membrane. We have used a baculovirus dual-expression system to produce various functionally complementing fragments of MRP1 in insect Sf21 cells to precisely define the region in CL3 that is required for activity and substrate binding. Using a parallel approach in polarized MDCK-I cells, we have also defined the region of CL3 that is required for basolateral trafficking. The CL3 NH(2)- and COOH-proximal functional boundaries have been identified as Cys(208) and Asn(260), respectively. Cys(208) also corresponds to the NH(2)-proximal boundary of the region required for basolateral trafficking in MDCK-I cells. However, additional residues downstream of the CL3 COOH-proximal functional boundary extending to Lys(270) were found to be important for basolateral localization. Finally, we show that regions in CL3 necessary for LTC(4) binding and transport are also required for binding of the photoactivatable GSH derivative azidophenacyl-GSH.

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

“…MRP1 expression vectors encoding proteins lacking the first 203 amino acid residues or with mutated/chimeric COOH-terminal regions have been described previously (Grant et al, 1994;Gao et al, 1996;Qian et al, 2001a;Westlake et al, 2003Westlake et al, , 2004. Vectors for proteins that contained both modified NH 2 -and COOH-terminal regions were generated by ligation of mutant MRP1 fragments from the above-mentioned vectors into an appropriate recipient vector.…”

“…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 have an extracellular NH 2 terminus (Hipfner et al, 1997;Kast and Gros, 1997;Tusnady et al, 1997;Raab-Graham et al, 1999). A similar third MSD also is present in the yeast ABCC protein cadmium factor Ycf1, a probable ortholog of MRP1 (Li et al, 1996), and in other ABCC proteins identified in plants and lower eukaryotes (Tusnady et al, 1997).In MRP1, MSD0 is connected to the remainder of the protein by a cytoplasmic loop, CL3, of ϳ108 amino acids (Westlake et al, 2003). The cytoplasmic NH 2 termini of ABCC proteins lacking MSD0 show some conservation of sequence with the start of CL3 in proteins such as MRP1, suggesting that the additional MSD may have been acquired by fusion of a gene encoding a core ancestral ABCC protein with one or more genes encoding other integral membrane proteins .…”

“…In contrast to SUR1A, MRP2, and Ycf1, MRP1 lacking MSD0 (MRP1 204 -1531 ) can traffic to the basolateral membrane in polarized cells and transport at least some substrates relatively efficiently (Bakos et al, 1998;Westlake et al, 2003). However, further truncation to remove as few as six amino acids from CL3 markedly decreases transport activity in insect cells and the protein is retained in the endoplasmic reticulum (ER) of mammalian cells (Westlake et al, 2003). NH 2 -terminal truncation of MRP1 to Ala 213 in CL3 severely disrupts substrate binding and transport even in insect cells (Westlake et al, 2003).…”

“…However, further truncation to remove as few as six amino acids from CL3 markedly decreases transport activity in insect cells and the protein is retained in the endoplasmic reticulum (ER) of mammalian cells (Westlake et al, 2003). NH 2 -terminal truncation of MRP1 to Ala 213 in CL3 severely disrupts substrate binding and transport even in insect cells (Westlake et al, 2003). Thus, although the integrity of the cytoplasmic loop immediately after MSD0 is essential for protein processing and activity, it has been concluded that MSD0 is dispensable, at least with respect to trafficking and transport of some substrates.…”

“…All other transient and stable transfectants were grown on glass coverslips coated with 0.1% gelatin. For subcellular localization studies, cells were fixed with 95% ice-cold ethanol, with the exception that 2% paraformaldehyde and digitonin (25 g/ml) were used with certain antibodies, as described previously (Westlake et al, 2003). Cells were incubated with previously characterized MRP1 mAbs (with the exception of MRP1 variants fused to CFP and YFP), with and without mAb anti-calnexin (Sigma Diagnostics Canada), polyclonal antibody (pAb) TGN46, or mAb Lamp-2 (H4B4) (Santa Cruz Biotechnology, Santa Cruz, CA).…”