Probing ATP-dependent conformational changes in the multidrug resistance protein 1 (MRP1/ABCC1) in live tumor cells with a novel recombinant single-chain Fv antibody targeted to the extracellular N-terminus

Abstract

The multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-driven transporter that mediates the cellular extrusion of various chemotherapeutic agents. We have previously isolated a novel recombinant single-chain Fv antibody (A5scFv), which specifically targets the extracellular N-terminus of the human MRP1 expressed on the surface of live tumor cells. Fusion of A5scFv to Pseudomonas exotoxin revealed an immunotoxin that bound to the immobilized MRP1-derived peptide upon ELISA, but surprisingly failed to recognize MRP1 on the surface of live tumor cells. As these results suggested that the N-terminus of MRP1 has a limited accessibility to the extracellular space, we used the A5scFv antibody to probe for putative conformational changes that might occur in viable tumor cells upon ATP binding. A5scFv recognized viable MRP1-expressing cells with intact ATP pools, whereas ATP depletion resulted in the loss of A5scFv reactivity. Consistently, restoration of cellular ATP levels resulted in resumption of A5scFv binding to MRP1 in live tumor cells. Flow cytometric analysis confirmed that ATP-depleted cells accumulated significantly higher levels of the established substrate calcein AM, whereas after restoration of cellular ATP pools, cells displayed a much lower level of calcein AM accumulation. Moreover, pretreatment of MRP1-expressing cells with the membrane fluidizer benzyl alcohol resulted in a dramatic increase in A5scFv reactivity, suggesting that membrane fluidization results in the exposure of the N-terminus of MRP1 to the extracellular milieu. These results constitute the first extracellular probing of the putative conformational changes that MRP1 adopts in viable tumor cells upon ATP binding. Furthermore, although ATP binding occurs in the cytosolic nucleotide binding domains of MRP1, significant conformational changes are apparently propagated to the N-terminus residing at the extracellular compartment. © 2005 Wiley-Liss, Inc.