ABCMdb

PMID: 15260487

Polgar O, Robey RW, Morisaki K, Dean M, Michejda C, Sauna ZE, Ambudkar SV, Tarasova N, Bates SE
Mutational analysis of ABCG2: role of the GXXXG motif.
Biochemistry. 2004 Jul 27;43(29):9448-56., 2004-07-27 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCG2 p.Arg482Gly All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. Login to comment 4 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Login to comment 5 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Login to comment 54 ABCG2 p.Arg482Gly This vector was selected for ease of study with rhodamine 123, a substrate for ABCG2 only when the R482G mutation is present. Login to comment 55 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Ala ABCG2 p.Gly410Ala ABCG2 p.Gly406Ala ABCG2 p.Gly406Ala The following ABCG2 mutants were generated: G406L, G410L, G406L/G410L, G406A, G410A, and G406A/ G410A. Login to comment 58 ABCG2 p.Gly410Leu MXR406/410 5' and MXR406/ 410 3' contain the mutation sites of amino acids 406 and 410 (glycine to leucine or alanine at either or both sites). Login to comment 124 ABCG2 p.Arg482Gly In the present study we used this R482G "gain-of-function" mutation in all of the mutants in order to be able to study them with flow cytometry using rhodamine 123 as a substrate. Login to comment 130 ABCG2 p.Arg482Gly When compared to the R482G transfectant clone, lower levels in most of the leucine mutants were consistently observed by immunoblot analysis. Login to comment 131 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu This was particularly true for the G406L/G410L double mutants. Login to comment 138 ABCG2 p.Arg482Gly Rhodamine 123 is transported by ABCG2 only when the R482G amino acid substitution is present. Login to comment 140 ABCG2 p.Arg482Gly Assessment of mitoxantrone accumulation revealed some transport in the same mutants, but activity was impaired when compared to the R482G transfected clones. Login to comment 141 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu As indicated by almost no change in the level of fluorescence for mitoxantrone in the G406L/G410L mutant, this mutation impairs mitoxantrone transport more than the single mutations. Login to comment 143 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu As shown in Figure 3 the double leucine mutant does not transport this compound, while the G406L and G410L mutants show impaired transport of pheophorbide a. Login to comment 145 ABCG2 p.Gly410Ala ABCG2 p.Gly406Ala Transport studies for the GXXXG alanine mutants using the same ABCG2 substrates were also performed; Figure 3 shows the results obtained with the G406A/G410A mutant. Login to comment 146 ABCG2 p.Arg482Gly ABCG2 p.Gly410Ala ABCG2 p.Gly406Ala Changes in fluorescence when FTC was added were almost identical to the R482G mutant, indicating that alanine is able to replace glycine without interfering with protein function. Flow cytometry also proved that the G406A and G410A mutants had intact transport of these substrates (data not shown). Login to comment 151 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Using DSS, which cross-links proteins 11.4 Å apart, a dimer or higher order multimer was observed by immunoblot analysis in the G406L and G406L/G410L mutants and in the R482G control cells, as shown in Figure 4A. Next, cross-linking studies were performed with DSP, an agent that can be cleaved with the addition of DTT or -mercaptoethanol. Login to comment 153 ABCG2 p.Arg482Gly (A) Protein expression levels on immunoblot with the BXP21 monoclonal anti-ABCG2 antibody for two clones of each of the leucine mutants (25 µg of protein per lane), in most cases revealing significantly lower levels than in the R482G clone, from which they were derived. Login to comment 155 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu (C) RNA levels of one representative clone of each of the leucine and alanine mutants on Northern blot with one of the highest levels in the G406L/G410L mutant. Login to comment 160 ABCG2 p.Arg482Gly Levels are compared to the R482G transfected cells. Plots: ABCG2 (---); negative control (s). Login to comment 161 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Ala ABCG2 p.Gly406Ala Transport capacity for BODIPY-prazosin, rhodamine 123, mitoxantrone, and pheohorbide A in the R482G, G406A/G410A, G406L/G410L, G406L, and G410L transfected cells. Plots: Accumulation without FTC (s) and with FTC (---). Login to comment 162 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Note lost BODIPY-prazosin and pheophorbide a transport with greatly impaired mitoxantrone transport in the double leucine mutant (G406L/G410L) and almost completely lost rhodamine 123 transport in all leucine mutants. Login to comment 163 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu The single leucine mutants (G406L, G410L) show impaired transport for BODIPY-prazosin, mitoxantrone, and pheophorbide a when compared to the fully functional R482G control cell line. Login to comment 164 ABCG2 p.Gly410Ala ABCG2 p.Gly406Ala The G406A/G410A mutants show intact transport capacity for all studied substrates. Login to comment 166 ABCG2 p.Arg482Gly (A) Cross-linking of ABCG2 is observed with DSS in HEK293 cells transfected with R482G or with the functionally impaired leucine mutants. Login to comment 167 ABCG2 p.Arg482Gly (B) Cross-linking is observed with the cleavable cross-linker DSP in the R482G mutant; note that the same higher molecular weight band can be seen under nonreducing conditions and that it can be reduced to the monomeric 72 kDa band with the addition of -ME or DTT. Login to comment 168 ABCG2 p.Gly410Leu ABCG2 p.Gly406Ala (C) Dimerization is suggested in both G406A and G410L mutants by high molecular weight bands detected following either exposure to DSP or harvesting under nonreducing conditions. Login to comment 170 ABCG2 p.Gly406Leu ABCG2 p.Gly410Ala (D) Similar results were obtained for the G406L and G410A mutants. Login to comment 172 ABCG2 p.Arg482Gly Cross-linking of the R482G transfected cells with DSP resulted in a high molecular weight protein, representing a dimer or multimer, which could be reduced to the monomeric 72 kDa form by exposure to reducing agents (Figure 4B). Login to comment 174 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Ala ABCG2 p.Gly410Ala ABCG2 p.Gly406Ala ABCG2 p.Gly406Ala Identical results were obtained with all the leucine and alanine mutants (Figure 4C showing results for the G406A and G410L mutants and Figure 4D for G406L and G410A; similar data not shown for the G406L/G410L and G406A/G410A mutants), implying that even if dimerization is impaired in these mutants, it has not prevented their close association on the cell surface. Login to comment 179 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu We determined the vanadate-sensitive component of the ATPase activity of the G406L/G410L mutant in the presence of 0, 1, 10, and 100 µM prazosin and compared it to the R482G and empty pcDNA3.1 vector transfected cells. Login to comment 180 ABCG2 p.Arg482Gly Figure 5 shows that 100 µM prazosin increased the ATP hydrolysis activity of the R482G transfected clone approximately 3-fold over basal level, while the empty vector transfected control cell line (pcDNA) had a significantly lower basal level with a negligible increase in activity in the presence of the drug. Login to comment 181 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Significantly, the G406L/G410L mutant, which previously showed the greatest impairment in transport capacity by flow cytometry (Figure 3), revealed ATPase levels identical to the empty vector transfected cell line with no stimulation with prazosin. Login to comment 185 ABCG2 p.Arg482Gly Cells were thus incubated in 2-10 µM mitoxantrone overnight, membranes harvested, and proteins separated by electrophoresis. Figure 6A shows a significant increase in protein levels on imunoblot for the GXXXG motif mutants, with a less pronounced increase in the cells expressing protein without this mutation (R482G), reaching a plateau at 5 µM mitoxantrone. Login to comment 186 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu The increased ABCG2 expression after overnight treatment with mitoxantrone in the G406L and G406L/G410L mutants was also confirmed with confocal microscopy (Figure 6B). Login to comment 192 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu While the double leucine mutant G406L/G410L was found on the cell surface, protein levels were reduced on immunoblot, and transport function and ATP hydrolysis were markedly impaired. Login to comment 201 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu The vanadate-sensitive ATP hydrolysis in the presence of the indicated concentrations of prazosin for crude membranes of HEK293 cells expressing ABCG2 R482G (0), G406L/G410L mutants (]), and empty vector transfected cells (pcDNA; O) is shown. Login to comment 202 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu The G406L/G410L mutant demonstrates basal ATPase levels identical to those of the pcDNA with no substrate induction observed. Login to comment 225 ABCG2 p.Arg482Gly Although the mutant proteins are located on the cell surface, they are expressed at a lower level than the R482G mutant, to which they should be compared. Login to comment 231 ABCG2 p.Arg482Gly (A) Overnight treatment of intact cells with 2-10 µM mitoxantrone substantially increases the level of ABCG2 in the GXXXG single and double leucine mutants (two clones for each shown) on immunoblot, while the change is much less pronounced in the control R482G transfected cells. Login to comment 232 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu (B) Confocal microscopy of HEK293 cells transfected with ABCG2 mutants shows a significant increase in ABCG2 levels for the G406L and G406L/G410L mutants after overnight treatment with 5 µM mitoxantrone. Login to comment 242 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Remarkably, both the basal and prazosin-stimulated ATPase activity was found to be severely impaired in the G406L/G410L mutant. Login to comment 247 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Since both the basal and the prazosin-stimulated ATP hydrolyses were impaired in the G406L/G410L mutant, it is conceivable that the GXXXG motif could play a similar role in ABCG2. Login to comment