Purification of breast cancer resistance protein ABCG2 and role of arginine-482

Abstract

Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding.

Role of Basic Residues within or near the Predicted Transmembrane Helix 2 of the Human Breast Cancer Resistance Protein in Drug Transport

“…The K m values of K453D, R465A, and K473A were comparable with that of wild-type protein; however, the K m values of K452A and H457A were decreased by approximately 50 and 70%, respectively, suggesting that these two mutations, particularly the one at position 457, increased the binding affinity of ATP to BCRP. The K m value of wild-type BCRP was comparable with that reported previously (Pozza et al, 2006). After normalization to the BCRP levels, the V max values of K453D and R465A were approximately 60% lower than that of wild-type BCRP, whereas the V max values of other mutants did not change.…”

“…For example, replacing a Pro residue in the third MSD of MRP1 with Ala caused an increase in transporting 17␤-estradiol 17␤-D-glucuronide that was associated with a 4-fold decrease in K m for ATP (Koike et al, 2004). More importantly, the K m and V max /K m values for basal ATPase activity of R482T with a similar gain of function have also been shown to be decreased and increased, respectively (Pozza et al, 2006). As to why mutations of these basic residues affect ATPase activity is not known.…”

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

“…These findings confirm that MDR3 NBDs are able to accomplish substrate-stimulated ATP hydrolysis in the detergent-solubilized state. Despite the high degree of amino acid sequence identity between MDR1 and MDR3 (Ͼ85% homology to human MDR1 and 80% to mouse Mdr1a (previously called mouse MDR3)), they exhibited different maximal ATPase activities in the presence of the transport substrate but similar and relatively high K m values for ATP (MDR3, K m ϭ 1.90 Ϯ 0.27 mM; MDR1, K m ϭ 1.5 mM (44)), which were in a range typical for ABC transporters (36,40,44,57). Because PC lipids are present in high concentrations in the plasma membrane, Ishigami et al (17) suggested that MDR3 is a low affinity transporter optimized for PC translocation.…”

ABCG2 Transports and Transfers Heme to Albumin through Its Large Extracellular Loop*

“…Finally, we measured the binding of nonporphyrin substrates of ABCG2, such as mitoxantrone (3) and riboflavin (56), and other drugs that bind to ABCG2 but are not transported, such as doxorubicin and rhodamine 123 (12). Results are displayed in Fig.…”

“…The whole ABCG2 transporter was expressed in insect cells, solubilized in ␤-D-dodecyl maltoside and purified as previously described (12,39) and detailed here under "Experimental Procedures." ABCG2 (0.1 M) was first incubated with 5 M mitoxantrone or doxorubicin, a concentration large enough to saturate the transports sites, based on the respective K d values of 0.9 and 1.6 M previously reported (12). Then, increasing concentrations of hemin (Fig.…”