Abstract

The human multidrug resistance protein 1 (MRP1) is a primary active transporter of reduced (GSH) and oxidized glutathione, as well as GSH-, glucuronate-, and sulfate-conjugated organic anions. In addition, the transport of certain MRP1 substrates is stimulated by the presence of GSH. To evaluate the structural features of GSH required for interaction with the protein, we investigated the ability of a series of GSH analogs to enhance GSH stimulatable transport of [³H]estrone 3-sulfate (E₁SO₄). We found that substitution of the γ-Glu residue with Gly, β-Asp, and α-Glu resulted in complete loss of transport stimulation. In contrast, substitution of Gly with Glu or β-Ala resulted in only a partial loss of stimulatory activity. E₁SO₄transport activity surpassed GSH-stimulated levels in the presence of tripeptides in which Cys was substituted with the hydrophobic amino acids Leu, Phe, and homo-Phe. Moreover, polar substitutions of Cys did not enhance transport to the same extent as nonpolar substitutions of comparable size. γ-Glu-Leu-Gly was 1.6-fold more effective than GSH in stimulating E₁SO₄ uptake, and kinetic analysis indicated this was due to an increasedV_max. In addition, this tripeptide was shown to be a competitive inhibitor of apigenin-stimulated GSH transport (K_i value of 14 μM), confirming that it either interacts with the same site on MRP1 as GSH or that the binding of the two tripeptides is mutually exclusive. These data provide insight into the architecture of the GSH binding domain of MRP1.