TY - JOUR

T1 - Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2

AU - Henriksen, Ulla Birk

AU - Gether, Ulrik

AU - Litman, Thomas

PY - 2005/4/1

Y1 - 2005/4/1

N2 - The ATP binding cassette (ABC) half-transporter ABCG2 (MXR/BCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. The mutation did not affect the predicted oligomerization properties of the transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABCG2-K86M. We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. This is consistent with the requirement for at least two active NBDs for transporter activity and suggests that the transporter is a functional dimer. Finally, we analyzed targeting of ABCG2-wt and ABCG2-K86M and observed that they localize to two distinct subcellular compartments: ABCG2-wt targets the cell surface whereas ABCG2-K86M is targeted to the Golgi apparatus followed by retrieval to the endoplasmic reticulum. This suggests an as yet unknown role of the NBDs in assisting proper surface targeting of ABC transporters.

AB - The ATP binding cassette (ABC) half-transporter ABCG2 (MXR/BCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. The mutation did not affect the predicted oligomerization properties of the transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABCG2-K86M. We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. This is consistent with the requirement for at least two active NBDs for transporter activity and suggests that the transporter is a functional dimer. Finally, we analyzed targeting of ABCG2-wt and ABCG2-K86M and observed that they localize to two distinct subcellular compartments: ABCG2-wt targets the cell surface whereas ABCG2-K86M is targeted to the Golgi apparatus followed by retrieval to the endoplasmic reticulum. This suggests an as yet unknown role of the NBDs in assisting proper surface targeting of ABC transporters.

KW - ATP-Binding Cassette Transporters

KW - Adenosine Triphosphatases

KW - Adenosine Triphosphate

KW - Amino Acid Substitution

KW - Cell Compartmentation

KW - Cell Line

KW - Cell Membrane

KW - Cell Survival

KW - Drug Resistance, Multiple

KW - Humans

KW - Hydrolysis

KW - Mitoxantrone

KW - Mutation

KW - Neoplasm Proteins

KW - Nucleotides

KW - Prazosin

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Protein Transport

U2 - 10.1242/jcs.01729

DO - 10.1242/jcs.01729

M3 - Journal article

C2 - 15769853

VL - 118

SP - 1417

EP - 1426

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - Pt 7

ER -