ABCMdb

PMID: 15557326

Ozvegy-Laczka C, Varady G, Koblos G, Ujhelly O, Cervenak J, Schuetz JD, Sorrentino BP, Koomen GJ, Varadi A, Nemet K, Sarkadi B
Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface.
J Biol Chem. 2005 Feb 11;280(6):4219-27. Epub 2004 Nov 22., 2005-02-11 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Lys86Met The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. Login to comment 8 ABCG2 p.Lys86Met In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Login to comment 53 ABCG2 p.Lys86Met Sf9 cells expressing the ABCG2 protein or its K86M variant were prepared as described previously (31). Login to comment 54 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met ABCG2 p.Lys86Met In the present study we used the K86M variant introduced into the wild-type (R482) ABCG2 by cloning the NotI-SpeI fragment of pAcUW21-L/K86M-R482G (31) into the corresponding site of the pAcUW21-L/R482 vector. Login to comment 83 ABCG2 p.Lys86Met ATPase Activity Measurement-Sf9 membranes containing human ABCG2, MDR1, or ABCG2-K86M were harvested, and their membranes were isolated and stored at -80 °C according to Sarkadi et al. (34, 35). Login to comment 92 ABCG2 p.Lys86Met Panel A shows expression of human, wild-type ABCG2, or the K86M-ABCG2 variant in isolated membranes of Sf9 insect cells (11). Login to comment 93 ABCG2 p.Lys86Met Panel B shows BXP-21 immunoreactions with cell lysates of PLB cells, engineered to express the wild-type ABCG2 or its K86M mutant variant. Login to comment 94 ABCG2 p.Lys86Met The expression level of the K86M variant of ABCG2 was about one-third the expression obtained for the wild-type protein (these cells could not be selected by mitoxantrone; see "Experimental Procedures" and Ozvegy et al. (31)). Login to comment 110 ABCG2 p.Lys86Met ABCG2 p.Lys86Met Sf9 membranes containing WT ABCG2, ABCG2-K86M, or beta-galactosidase (beta-gal, panel A) and cell lysates from PLB (panel B), HEK 293 (panel C), and MCF-7/MX (panel D) cells expressing WT ABCG2 (or ABCG2-K86M) or parental cells (ctr.) Login to comment 131 ABCG2 p.Lys86Met There was no effect of 5D3 antibody on the ATPase activity of MDR1 or ABCG2-K86M membranes. Login to comment 146 ABCG2 p.Lys86Met Sf9 membranes containing WT ABCG2, ABCG2-K86M, or MDR1 were incubated with 20 (low 5D3, hatched columns) or 160 ␮g (high 5D3, black columns)/mg membrane concentration of 5D3 antibody. Login to comment 161 ABCG2 p.Arg482Gly We obtained essentially similar data in the MCF-7/MX cells and the PLBs expressing the gain-of-function R482G mutant of ABCG2 (not shown). Login to comment 182 ABCG2 p.Lys86Met ABCG2 p.Lys86Met Effects of Substrates, Inhibitors, and ATP Depletion on 5D3 Reactivity in the Mutant, Non-functional K86M-ABCG2, Expressed in Intact Cells-In the next set of experiments we studied intact mammalian cells expressing a non-functional mutant (K86M) variant of ABCG2. Login to comment 184 ABCG2 p.Lys86Met As shown in Fig. 6, panels A and B, this K86M-ABCG2 had no MX extrusion function but showed a well measurable 5D3 binding on the cell surface. Login to comment 185 ABCG2 p.Lys86Met In these studies we found that the 5D3 binding of the K86M mutant ABCG2 was significantly increased by PFA fixation, ATP depletion, or Ko143 treatment. Login to comment 187 ABCG2 p.Lys86Met Thus, the non-functional K86M variant of ABCG2 showed a relatively high 5D3 binding in its native state, but in the case of ATP removal and Ko143 treatment, similar conformational changes were detected by 5D3 in this mutant variant as in the wild-type protein. Login to comment 188 ABCG2 p.Lys86Met The lack of the formation of a transition-state intermediate in the K86M-ABCG2 correlated with the absence of an effect of sodium orthovanadate. Login to comment 189 ABCG2 p.Lys86Met Effects of Nucleotides and Transport Inhibitors on 5D3 Reactivity of ABCG2 in Isolated Membrane Fragments-In the following experiments we examined the effects of various nucleotides and transport inhibitors on 5D3 binding by human ABCG2 and its mutant (K86M) variant in isolated insect cell membrane fragments. Login to comment 194 ABCG2 p.Lys86Met Fig. 7, B and C, documents the effects of various ligands on 5D3 binding to wild-type (panel B) or K86M (panel C) ABCG2 in isolated membranes. Login to comment 209 ABCG2 p.Lys86Met Fig. 7, panel C, shows 5D3 binding in isolated membranes containing the K86M, non-functional mutant ABCG2 protein. Login to comment 213 ABCG2 p.Lys86Met These data can be interpreted to mean that although MgAMP does not show binding to the protein, MgATP, MgADP, and MgAMP-PNP are bound to K86M-ABCG2, and in the absence of a full catalytic cycle, they fix the transporter in a nucleotide-bound, reduced 5D3 binding state. This fixation does not require the presence of vanadate. Login to comment 215 ABCG2 p.Lys86Met Interestingly, Ko143 can still stabilize the K86M-ABCG2 variant in a high 5D3 binding state. Login to comment 220 ABCG2 p.Lys86Met These effects were similar both in the wild-type ABCG2 and the K86M mutant variant (not documented in detail). Login to comment 237 ABCG2 p.Lys86Met In the present experiments the arrest of the ABCG2 transport cycle by Ko143 by the removal of the energy donor substrate, ATP, as well as by sodium orthovanadate was documented by the lack of active MX extrusion in the FIG. 6. Flow cytometry detection of the K86M-ABCG2 protein. Login to comment 238 ABCG2 p.Lys86Met 5D3 mAb binding (panel A) and MX extrusion (panel B) in K86M mutant ABCG2-expressing intact PLB cells is shown. Login to comment 250 ABCG2 p.Lys86Met When examining the binding of the 5D3 antibody in intact cells to a non-functional ABCG2 catalytic center mutant (K86M-ABCG2), we found that 5D3 binding to this mutant protein was also efficient. Login to comment 259 ABCG2 p.Lys86Met In the K86M-ABCG2 variant the addition of MgATP, MgADP, and MgAMP-PNP all caused a major reduction of 5D3 binding, which was not further modulated by sodium orthovanadate. Login to comment 263 ABCG2 p.Lys86Met Interestingly, in Sf9 membranes containing either wild-type ABCG2 or its K86M mutant in the absence of Mg2ϩ (that is, in the presence of EDTA), ATP, ADP, and AMP-PNP caused a decrease in 5D3 binding (not shown in detail). Login to comment 270 ABCG2 p.Lys86Met Isolated membrane fragments (45 ␮g) from Sf9 cells containing WT ABCG2, ABCG2-K86M, or MDR1 were labeled with 1 ␮g/ml 5D3 (black columns) or 1 ␮g/ml mouse IgG2b as isotype control (white columns). Login to comment 273 ABCG2 p.Lys86Met Panel C, 5D3 binding to membranes containing K86M mutant ABCG2. Login to comment 274 ABCG2 p.Lys86Met Sf9 membranes containing wild-type ABCG2 (panel B) or K86M mutant ABCG2 (panels C) were incubated with 5D3 antibody in the presence of 10 mM MgAMP, MgADP, MgATP, MgAMP-PNP, MgAMP plus 2 mM vanadate, MgATP plus 2 mM vanadate, MgATP plus 2 mM vanadate plus 1 ␮M Ko143, MgADP plus 1 ␮M Ko143, MgATP plus 1 ␮M Ko143, or 10 mM MgAMP-PNP plus 1 ␮M Ko143. Login to comment