ABCMdb

PMID: 15769853

Henriksen U, Gether U, Litman T
Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2.
J Cell Sci. 2005 Apr 1;118(Pt 7):1417-26. Epub 2005 Mar 15., 2005-04-01 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly It should be noted that an amino acid substitution at position 482 distinguishes MXR (R482G), BCRP (R482T) and ABCP (R482, wt) (Allikmets et al., 1998; Doyle et al., 1998; Miyake et al., 1999), which are synonymous designations for ABCG2. Login to comment 15 ABCG2 p.Lys86Met The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. Login to comment 16 ABCG2 p.Lys86Met 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. Login to comment 17 ABCG2 p.Lys86Met We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Login to comment 18 ABCG2 p.Lys86Met Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. Login to comment 20 ABCG2 p.Lys86Met ABCG2 p.Lys86Met 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. Login to comment 22 ABCG2 p.Lys86Met Key words: ABC transporters, ABCG2, Oligomerization, Trafficking, Multidrug resistance, Walker A mutation Summary Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2 Ulla Henriksen1 , Ulrik Gether1 and Thomas Litman2, * 1 Molecular Neuropharmacology Group, Department of Pharmacology, The Panum Institute, Blegdamsvej 3, University of Copenhagen, DK-2200 Copenhagen, Denmark 2 Bioinformatics Centre, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark *Author for correspondence (e-mail: tlitman@binf.ku.dk) Accepted 13 January 2005 Journal of Cell Science 118, 1417-1426 Published by The Company of Biologists 2005 doi:10.1242/jcs.01729 Research Article segments. Login to comment 28 ABCG2 p.Lys86Met In agreement, we find that the mutant (ABCG2-K86M) is inactive. Login to comment 36 ABCG2 p.Lys86Met Construction of mutation and tags The ABCG2-K86M mutation was generated by a two-generation PCR technique using the Pfu polymerase (Stratagene, La Jolla, CA). Login to comment 38 ABCG2 p.Lys86Met ABCG2-wt was tagged with either the MYC epitope or the HA epitope whereas ABCG2-K86M was tagged with the HA epitope. Login to comment 89 ABCG2 p.Lys86Met Results To obtain a loss-of-function mutant of ABCG2 we mutated the conserved Walker A lysine to methionine (K86M). Login to comment 90 ABCG2 p.Lys86Met The ABCG2-K86M mutant was tagged at the N-terminus with the hemagglutinin (HA) epitope whereas ABCG2-wt was tagged with a MYC epitope. Login to comment 93 ABCG2 p.Lys86Met The epitope tags did not alter apparent expression; however, the expression levels of the K86M mutants were somewhat lower than those observed for the wild-type constructs (Fig. 1A). Login to comment 94 ABCG2 p.Lys86Met The activity of ABCG2-K86M in comparison to ABCG2-wt was first assessed by measurement of ATPase activity. Login to comment 95 ABCG2 p.Lys86Met Whereas the ABCG2-wt membrane fraction showed dose-dependent and saturatable stimulation of ATPase activity in response to increasing concentrations of the substrate prazosin (EC50=3.5 µM; Vmax=7.6 nmol/minute/mg protein), no stimulation of the ATPase activity was detected in membranes from cells expressing ABCG2-K86M and the basal ATPase activity was comparable to that of the empty HEK293 cells (Fig. 1B). Login to comment 98 ABCG2 p.Lys86Met ABCG2 p.Lys86Met In contrast, ABCG2-K86M and ABCG2-K86M-HA displayed sensitivity comparable to that of non-transfected cells consistent with loss of function with IC50 values for mitoxantrone of 0.047 µM and 0.043 µM, respectively (Fig. 1C). Login to comment 100 ABCG2 p.Lys86Met These results further confirmed that ABCG2-K86M was non-functional. Login to comment 101 ABCG2 p.Lys86Met ABCG2 p.Lys86Met Cells expressing ABCG2-wt and ABCG2-wt-MYC efficiently expelled the substrate; however, this was not the case for cells expressing ABCG2-K86M or ABCG2-K86M-HA, which displayed transport activity comparable to non-transfected cells (Fig. 2). Login to comment 104 ABCG2 p.Lys86Met We wanted to further test this hypothesis and also analyze whether the K86M mutation altered the oligomerization properties of ABCG2. Login to comment 105 ABCG2 p.Lys86Met Accordingly, we coexpressed ABCG2-wt-MYC with ABCG2-K86M-HA as well as we coexpressed ABCG2-wt-MYC with ABCG2-wt tagged with HA instead of MYC. Login to comment 108 ABCG2 p.Lys86Met Both ABCG2-wt-HA and ABCG2-K86M-HA co-immunoprecipitated with ABCG2-wt-MYC (Fig. 3, lanes 1,2). Login to comment 111 ABCG2 p.Lys86Met However, we did not see the difference between the cell lines as a general phenomenon in our immunoprecipitations and in all our other experiments equal amounts of wt-HA and K86M-HA were precipitated (data not shown). Login to comment 114 ABCG2 p.Lys86Met Several controls were included to exclude non-specific interactions in the co-immunoprecipitation assay; cells transfected with either ABCG2-wt-MYC or ABCG2-K86M-HA showed no crossreactivity between the two tags (Fig. 3, lanes 3,4). Login to comment 119 ABCG2 p.Lys86Met The K86M mutation in ABCG2 results in a non-functional transporter. Login to comment 120 ABCG2 p.Lys86Met ABCG2 p.Lys86Met (A) Protein expression analyzed on isolated membrane fractions from control (empty) HEK293 cells, or HEK293 cells stably expressing ABCG2-wt, ABCG2-wt-MYC (ABCG2-wt-cmyc), ABCG2-K86M and ABCG2-K86M-HA. Login to comment 124 ABCG2 p.Lys86Met Vanadate-sensitive drug stimulated ATPase activity (mean±s.e.m., n=6) was measured with increasing concentrations of prazosin (0-50 µM) on ABCG2-wt, ABCG2-K86M and empty HEK293 cells. Login to comment 128 ABCG2 p.Lys86Met ABCG2 p.Lys86Met Sulforhodamine B staining was used to detect survival of ABCG2-wt, ABCG2-wt-MYC (ABCG2-wt-cmyc), ABCG2-K86M, ABCG2-K86M-HA and empty HEK293 cells. Login to comment 130 ABCG2 p.Lys86Met ABCG2 p.Lys86Met HEK293 empty ABCG2-wt-myc ABCG2-K86M ABCG2-K86M-HAABCG2-wt Fig. 2. Login to comment 131 ABCG2 p.Lys86Met HEK293 cells transfected with ABCG2-K86M display no transport activity. Login to comment 138 ABCG2 p.Lys86Met Altogether, this suggests that both ABCG2-wt and ABCG2-K86M form disulfide-bridge linked dimers. Login to comment 143 ABCG2 p.Lys86Met We examined this by ATPase activity measurements on isolated membrane fractions from cells coexpressing ABCG2-wt-MYC and ABCG2-K86M-HA, cells coexpressing ABCG2-wt-MYC and ABCG2-wt-HA, or cells expressing ABCG2-wt alone. Login to comment 145 ABCG2 p.Lys86Met We also found based on densitometry analysis of several western blots that the co-transfected cells expressed equal amounts of HA-tagged transporter (ABCG2-K86M-HA or ABCG2-wt-HA) and MYC-tagged transporter (ABCG2-wt-MYC) (Fig. 5B). Login to comment 148 ABCG2 p.Lys86Met However, in cells coexpressing ABCG2-wt-MYC and ABCG2-K86M-HA we found a markedly lower Vmax both compared to cells expressing ABCG2-wt alone and to cells coexpressing ABCG2-wt-MYC and ABCG2-wt-HA (EC50= 3.8±0.9 µM; Vmax=5.2±0.1 nmol/minute/mg protein; P<0.0001, unpaired t-test, Vmax compared to either ABCG2-wt alone or ABCG2-wt-MYC/ABCG2-wt-HA). Login to comment 149 ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Lys86Met The decrease in activity upon co-transfection of ABCG2-wt and ABCG2-K86M is consistent with a dominant-negative effect caused by the formation of an inactive wt/K86M complex and, accordingly, supports the fact that ABCG2 is a functional dimer. We should note, however, that based on the dominant-negative effect observed, we cannot exclude the fact that the transporters may exist and function as a higher order form than a dimer. We also investigated the subcellular localization of ABCG2-wt in comparison to ABCG2-K86M. Login to comment 150 ABCG2 p.Lys86Met Interestingly, immunocytochemical studies showed a distinct staining pattern of ABCG2-wt in comparison to ABCG2-K86M. Login to comment 152 ABCG2 p.Lys86Met In contrast, ABCG2-K86M staining was punctuate, and found almost exclusively in an intracellular compartment (Fig. 6D,E); hence, mutation of Lys86 might not only affect the functional properties of the transporter but also its cellular targeting. Login to comment 153 ABCG2 p.Lys86Met In order to obtain a quantitative measurement for the decrease in surface expression of the K86M mutation compared to ABCG2-wt, the cell lines were Fig. 3. Login to comment 154 ABCG2 p.Lys86Met ABCG2-wt and ABCG2-K86M co-immunoprecipitate. Login to comment 169 ABCG2 p.Lys86Met The experiments showed that the apparent surface expression of ABCG2-K86M was ~30% of that observed for the wild type consistent with our immunofluorescence data (Fig. 7A). Login to comment 170 ABCG2 p.Lys86Met In cells coexpressing ABCG2-wt-MYC and ABCG2-wt-HA or ABCG2-wt-MYC and ABCG2-K86M-HA we observed that the apparent surface expression in both cases was 30-40% higher than in cells only expressing ABCG2-wt-MYC. Login to comment 172 ABCG2 p.Lys86Met ABCG2 p.Lys86Met This was supported by immunostaining cells expressing K86M alone in comparison to cells expressing both K86M-HA and wt-MYC. Login to comment 173 ABCG2 p.Lys86Met ABCG2 p.Lys86Met As shown in Fig. 7B the vast majority of anti-HA staining was found intracellularly in cells expressing only K86M-HA whereas in the cells expressing K86M-HA together with wt-MYC we observed increased plasma membrane anti-HA staining. Login to comment 174 ABCG2 p.Lys86Met To determine the subcellular compartment in which ABCG2-K86M was localized we performed a series of co-stainings using the anti-ABCG2 antibody together with markers for different cellular compartments. Login to comment 175 ABCG2 p.Lys86Met We found similar staining patterns for ABCG2-K86M and anti-calnexin, a marker for the endoplasmic reticulum (ER) (Fig. 8B). Login to comment 177 ABCG2 p.Lys86Met Altogether, we conclude that ABCG2-K86M is mainly localized to the ER although a small percentage is localized to the plasma membrane. Login to comment 178 ABCG2 p.Lys86Met To verify our localization results we analyzed the glycosylation state of ABCG2-wt and ABCG2-K86M. Login to comment 196 ABCG2 p.Lys86Met These data imply that ABCG2-K86M is processed beyond the ER and possibly to cis-Golgi from where it is retrieved again to the ER where it is mainly detected. Login to comment 198 ABCG2 p.Lys86Met Furthermore, we show that mutation to methionine of the highly conserved lysine in Walker A (K86M) not only results in a non-functional transporter but also has a profound effect on targeting of the transporter to the cell surface. Login to comment 203 ABCG2 p.Lys86Met In ABCG2, it has been demonstrated by expression in Sf-9 insect cells that K86M is functionally inactive but still capable of binding ATP (Ozvegy et al., 2002). Login to comment 204 ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Lys86Met Accordingly, we chose K86M to address whether ABCG2 is a functional oligomer and established co-transfected stable HEK293 cell lines expressing the wild type alone, K86M alone or wild type and K86M half-transporter together. Login to comment 205 ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Lys86Met Analysis of the cotransfected cells with respect to ATPase activity suggested a dominant-negative activity of the K86M mutant on wild-type activity, i.e. membranes from cells coexpressing wt-MYC and K86M-HA displayed less ATPase activity as compared to cells expressing the wild type alone and only about 40% of the ATPase activity was observed in cells expressing both wt-HA and wt-MYC (Fig. 5). Login to comment 207 ABCG2 p.Lys86Met ABCG2-K86M is localized in an intracellular compartment. Login to comment 211 ABCG2 p.Lys86Met ABCG2 p.Lys86Met (A) ABCG2-wt, (B) ABCG2-wt-HA, (C) ABCG2-wt-MYC (ABCG2-wt-cmyc), (D) ABCG2-K86M and (E) ABCG2-K86M-HA. Login to comment 213 ABCG2 p.Lys86Met ABCG2-K86M shows a markedly reduced surface expression. Login to comment 219 ABCG2 p.Lys86Met ABCG2 p.Lys86Met (B) HEK293 cells stably expressing ABCG2-K86M or ABCG2-wt-MYC + ABCG2-K86M-HA were analyzed by immunocytochemistry to detect localization of the transporter. Login to comment 223 ABCG2 p.Lys86Met One possible explanation for this apparent discrepancy is that we have used stably transfected pool clones and accordingly cannot assume that the wild type and K86M are expressed in exactly equal amount in all cells. Login to comment 224 ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Lys86Met It is also possible that because the wild type and K86M preferentially are targeted to the ER and cell surface, respectively, we might not obtain the ideal distribution of 50% wt/K86M dimers, 25% wt/wt dimers and 25% K86M/ K86M. Login to comment 225 ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Lys86Met The presence of a dominant-negative effect supports the idea that the inactive K86M mutant is capable of oligomerizing with the wild type resulting in a nonfunctional wt/K86M transporter protein complex and, thus, that at least two functional NBDs are necessary for proper ATP hydrolysis. Login to comment 226 ABCG2 p.Leu554Pro Interestingly, a dominant-negative effect on drug sensitivity has been reported for ABCG2 (Kage et al., 2002) upon mutation of Leu554 situated in transmembrane segment 5 to a proline (L554P). Login to comment 231 ABCG2 p.Lys86Met The ABCG2-K86M mutant resides in the ER. Login to comment 238 ABCG2 p.Lys86Met ABCG2-K86M is glycosylated to the same degree as ABCG2-wt. Login to comment 240 ABCG2 p.Lys86Met Total cell lysates of ABCG2-wt or ABCG2-K86M were treated with denaturation buffer for 10 minutes at 100°C. Login to comment 248 ABCG2 p.Lys86Met We also assessed the oligomerization properties of ABCG2-wt and ABCG2-K86M by co-immunoprecipitation. Login to comment 259 ABCG2 p.Lys86Met Surprisingly, we observed a striking difference between the localization of ABCG2-wt (plasma membrane) and ABCG2-K86M (ER). Login to comment 261 ABCG2 p.Lys86Met A previous study on ABCG2-K86M suggested normal surface expression in Sf9 insect cells (Ozvegy et al., 2002). Login to comment 263 ABCG2 p.Lys86Met Immunocytochemical staining suggests that the K86M mutant resides in the ER, either because of retention or retrieval of the transporter to this compartment. Login to comment 264 ABCG2 p.Lys86Met In this context it is interesting to note that western blot analysis comparing the wild type and K86M showed that they both migrate identically in the gel. Login to comment 266 ABCG2 p.Lys86Met The glycosylation was, however, insensitive to Endo H indicating that both ABCG2-wt and ABCG2-K86M has been processed beyond the ER. Login to comment 267 ABCG2 p.Lys86Met This supports a scenario in which the K86M mutant is processed to the Golgi or the intermediary compartment and subsequently retrieved to the ER as part of a putative quality control mechanism. Login to comment 268 ABCG2 p.Lys86Met The available structural data on ABC transporters gives rise to speculation regarding how the K86M mutation affects surface targeting. Login to comment 271 ABCG2 p.Lys86Met As it was previously shown that the ABCG2-K86M mutation can still bind but not hydrolyze ATP (Ozvegy et al., 2002), a conceivable scenario might be that hydrolysis of ATP facilitates a closed structure of the NBDs; hence, a transporter unable to hydrolyse ATP is likely to have a markedly looser structure. Login to comment 272 ABCG2 p.Lys86Met Since the K86M mutation directly affects the ATP binding site, the NBD dimerization interface could be affected leading to impaired surface targeting followed by retrieval to ER. Login to comment 273 ABCG2 p.Lys86Met We still observe dimerization of the transporter when mutating K86M, suggesting that dimer formation is likely to be dependent on the transmembrane domains as well as on disulfide bridges formed between cysteines present in the predicted extracellular loops. Login to comment