ABCMdb

PMID: 19406100

Polgar O, Ediriwickrema LS, Robey RW, Sharma A, Hegde RS, Li Y, Xia D, Ward Y, Dean M, Ozvegy-Laczka C, Sarkadi B, Bates SE
Arginine 383 is a crucial residue in ABCG2 biogenesis.
Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Login to comment 5 ABCG2 p.Arg383Ala Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Login to comment 6 ABCG2 p.Arg383Ala Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. Login to comment 7 ABCG2 p.Arg383Ala The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. Login to comment 41 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly ABCG2 p.Arg383Gly ABCG2 p.Arg383Lys ABCG2 p.Arg383His ABCG2 p.Ser384Arg Mutagenesis and transfection The R383A, R383G, R383H, R383K, and R383G/S384R mutants were generated by site-directed mutagenesis in the pcDNA3.1/Myc-HisA(-) vector (Invitrogen) as previously described [26]. Login to comment 46 ABCG2 p.Arg482Gly Cells previously transfected with wild-type ABCG2, R482G and pcDNA vector only were used as controls [27]. Login to comment 66 ABCG2 p.Arg383Ala PCR-amplified wild-type ABCG2 or the R383A mutant from the appropriate pcDNA3.1 vectors were used as templates. Login to comment 80 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly Generation of Sf9 cells expressing the R383A and R383G mutants Generation of transfer vectors containing wild-type ABCG2 has been described previously [30,31]. Login to comment 81 ABCG2 p.Arg383Ala ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly ABCG2 p.Arg383Gly The transfer vectors carrying the R383A and R383G mutants were generated by cloning the SacI fragment of pcDNA 3.1/R383A or R383G into the corresponding site of the pAcUW21-L vector. Login to comment 93 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly ATP hydrolysis Sf9 membranes containing wild-type ABCG2, R383A, or R383G were harvested, and membranes were isolated and stored at -80 °C according to the method of Sarkadi et al. [33]. Login to comment 100 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly To begin investigating the role of arginine 383 in ABCG2, HEK 293 cells were stably transfected with pcDNA3.1 vectors carrying the R383G and R383A mutants. Login to comment 104 ABCG2 p.Arg383Ala First, flow cytometry performed on non-permeabilized cells using the 5D3 monoclonal antibody to recognize an extracellular epitope of ABCG2 demonstrated detectable levels of surface expression for some of the R383A clones, of which clones #11 and #24 exhibited the highest levels, though significantly lower compared to the wild-type (Fig. 2A). Login to comment 106 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly To test the functionality of the R383A and R383G mutants, flow cytometry was performed after incubating cells with the ABCG2-substrates mitoxantrone and pheophorbide a, with or without the ABCG2-inhibitor FTC (Fig. 2B). Login to comment 108 ABCG2 p.Arg383Ala The experiments were repeated several times, with limited efflux seen only in the R383A mutant as represented by a slight shift between the FTC-treated and non-treated histograms. Login to comment 110 ABCG2 p.Arg383Gly The R383G mutant, as expected from its absence at the cell surface, did not display transport activity for either mitoxantrone or pheophorbide a. Login to comment 111 ABCG2 p.Arg383Ala Protein expression levels on immunoblot with the BXP-21 monoclonal anti-ABCG2 antibody were significantly decreased for the R383A mutant, and a band slightly lower than the expected 72 kDa was also visible (Fig. 2C). Login to comment 113 ABCG2 p.Arg383Gly The R383G mutant clones were virtually not detectable, only clone #22 displayed some level of expression and was also represented by a double band (Fig. 2C). Login to comment 118 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly Finally, Northern blotting was carried out to confirm that the reduced expression of these mutants was not due to poor transfection efficacy; significant amounts of ABCG2 RNA were noted in representative clones of the R383A and R383G mutants (Fig. 2E). Login to comment 120 ABCG2 p.Arg383Ala These experiments showed no difference between wild-type ABCG2 and the R383A mutant, suggesting that, similarly to the wild-type, the mutant protein is translated and inserted properly into the membrane in the in vitro system and implying that the ER quality control must promote rapid degradation in vivo (data not shown). Login to comment 126 ABCG2 p.Arg383Ala To investigate the mechanisms leading to the dramatic decrease in protein levels observed with the mutants, the R383A mutant was incubated overnight separately with either the lysosome inhibitor bafilomycin, or the proteasome inhibitor MG132 (Fig. 3). Login to comment 128 ABCG2 p.Arg383Ala On the other hand, treatment with MG132 led to a 3 to 5-fold increase in the amount of the R383A mutant observed on immunoblot (Fig. 4A), indicating that the mutant is degraded by the ubiquitin-proteasome pathway. Login to comment 129 ABCG2 p.Arg383Ala To further explore the ER quality control mechanism behind the degradation of the mutant, we incubated the R383A mutant overnight in kifunensine, a potent inhibitor of mannosidase I. Mannose trimming by mannosidase I is one of the known events leading to ubiquitination and proteasomal degradation via the so-called glycan-dependent pathway (Fig. 3) [37]. Login to comment 132 ABCG2 p.Arg383Ala Overnight treatment with MG132 and kifunensine was also performed on HeLa cells transiently transfected with the wild-type and the R383A mutant providing results identical to the ones presented with the stable HEK transfectants (data not shown). Login to comment 137 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly Surface expression, function, protein and RNA levels of the R383A and R383G mutants transfected into HEK 293 cells. Login to comment 143 ABCG2 p.Arg383Ala (D) Immunoblot analysis of membrane proteins from wild-type (50 μg) and R383A (150 μg) transfectants with the BXP-21 following overnight treatment with endo H, or with N-glycosidase F. Login to comment 149 ABCG2 p.Arg383Ala Proteasome/lysosome inhibition and "rescue" of the R383A mutant with mitoxantrone (MX). Login to comment 150 ABCG2 p.Arg383Ala Parts A, B, and C: membranes were harvested subsequent to overnight incubation with or without 3 μM MG132, or 10 nM bafilomycin (part A), or 30 μg/mL kifunensine (part B), or 5 μM mitoxantrone (part C) followed by immunoblot analysis with the BXP-21 antibody for the wild-type (10 μg/lane) and R383A (10 μg/lane) transfectants. Login to comment 159 ABCG2 p.Arg383Ala Interestingly, in the case of the R383A mutant, though the overnight treatment with mitoxantrone did not result in increased protein expression level, a shift to the 72-kDa band was visible in the drug-treated lanes, suggesting that the drug did act as a chaperone and helped generate the mature, N-glycosylated form of the protein, although levels were not increased (Fig. 4C). Login to comment 162 ABCG2 p.Arg383Ala Localization of the R383A mutant in HEK 293 cells. Login to comment 166 ABCG2 p.Arg383Ala The R383A mutant in Sf9 insect cells. Login to comment 169 ABCG2 p.Lys86Met ABCG2 p.Arg383Ala (C) Basal ATPase activity of the wild-type, a 1:5 dilution of the wild-type, the R383A mutant, and the non-functional K86M mutant in Sf9 membranes. Login to comment 172 ABCG2 p.Arg383Ala While we previously observed an increase in the amount of the R383A mutant protein represented by the lower than 72-kDa band on immunoblot (Fig. 4A), we did not detect any increase on the cell surface with the 5D3 antibody, suggesting that by blocking the proteasomal degradation pathway the mutant protein accumulated intracellularly. Login to comment 173 ABCG2 p.Arg383Ala On the other hand, in agreement with the shift observed in the case of the R383A mutant to the 72-kDa mature form on immunoblot (Fig. 4C), we detected increased cell surface expression of the mutant protein by flow cytometry following overnight treatment with mitoxantrone (Fig. 4D). Login to comment 174 ABCG2 p.Arg383Gly Treatment with mitoxantrone resulted in a similar increase in the amount of the R383G mutant detected on the cell surface (data not shown). Login to comment 175 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly To further analyze the localization of the R383A and R383G mutants in the mammalian cells, immunofluorescent staining followed by confocal microscopy was performed. Login to comment 176 ABCG2 p.Arg383Ala In the case of the R383A mutant, colocalization with the ER marker calnexin was suggested, while some of this mutant also localized to the cell surface (Fig. 5), which is in agreement with the results of flow cytometry shown in Fig. 2. Login to comment 177 ABCG2 p.Arg383Gly The expression level for the R383G mutant was too low to determine localization using confocal microscopy. Login to comment 178 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly Next, we evaluated the R383G and R383A mutations in a heterologous system, using Sf9 insect cells, a transfection system that generally yields high protein levels allowing the study of proteins with low expression levels in mammalian cells [40]. Login to comment 180 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly Flow cytometry with the 5D3 monoclonal anti-ABCG2 antibody revealed that, similar to the mammalian cells, the R383A mutant was detectable on the surface in the insect cells, while the R383G mutant was not (Fig. 6B). Login to comment 183 ABCG2 p.Arg383Gly ABCG2 p.Arg383Lys ABCG2 p.Ser384Arg We postulated that this arginine might function as an anchor of the first transmembrane alpha helix, thus more conservative substitutions were performed: mutation to the positively charged lysine and introduction of arginine at position 384 instead of 383, creating the R383K and R383G/S384R mutants, followed by stable transfections in HEK 293 cells. Login to comment 184 ABCG2 p.Arg383Ala ABCG2 p.Arg383Lys Like R383A, the R383K mutant displayed some surface expression on flow cytometry (Fig. 7A) and the protein was detectable on immunoblot, though expression levels were still markedly reduced when compared to the wild-type transfectant (Fig. 7B). Login to comment 185 ABCG2 p.Arg383Lys Just as observed for the alanine mutant, R383K was represented by a double band on immunoblot suggestive of altered glycosylation. Login to comment 187 ABCG2 p.Arg383Lys Surface expression, function and protein levels of the R383K mutant transfected into HEK 293 cells. Login to comment 188 ABCG2 p.Arg383Lys (A) The R383K mutant (clone #8) detected on the cell surface with the 5D3 antibody by flow cytometry performed as described for Fig. 2. Login to comment 193 ABCG2 p.Arg383Lys functionality of the R383K mutant was also assessed in flow cytometry-based transport assays. Login to comment 195 ABCG2 p.Arg383Gly ABCG2 p.Ser384Arg On the other hand, none of the R383G/S384R mutant clones was detectable either on the cell surface by flow cytometry with the 5D3 antibody or on immunoblot with the BXP-21 antibody (data not shown). Login to comment 197 ABCG8 p.Arg405His Interestingly, we noted that an arginine to histidine mutation in the corresponding residue in ABCG8 (R405H) had been reported in a patient with sitosterolemia [41]. Login to comment 198 ABCG2 p.Arg383His To study the effect of the same substitution in ABCG2, we created stable R383H clones in HEK 293 cells. Login to comment 206 ABCG2 p.Arg383Ala ABCG2 p.Arg383Gly In this system mutating arginine 383 to glycine, alanine, histidine or lysine resulted in markedly reduced to no protein expression, impaired glycosylation and retention in the ER. Login to comment 207 ABCG2 p.Arg383Ala The R383A mutant was shown to be degraded by the proteasome via a kifunensine-insensitive pathway. Login to comment 223 ABCC1 p.Arg1249Ala For example, the R1249A mutant of MRP1 has been described as having altered substrate specificity. Login to comment 228 ABCG2 p.Arg383Ala Our results with the proteasome inhibitor MG132 indicate that the R383A mutant is degraded via the proteasome, while the wild-type protein is not. Login to comment 231 ABCG2 p.Arg383Ala The quality control pathway by which the R383A mutant is targeted to the proteasome seems to be kifunensine-insensitive. Login to comment 236 ABCG2 p.Arg383Ala Interestingly, in the case of the R383A mutant, mitoxantrone seemed to help generate the mature, glycosylated protein. Login to comment 240 ABCG2 p.Arg383Ala The R383A mutant localized to the cell surface and displayed some function in the HEK cells. Login to comment 241 ABCG2 p.Arg383Ala When transfected to Sf9 insect cells, R383A was also detected on the surface, yet was unable to hydrolyze ATP. Login to comment 253 ABCG2 p.Arg383Gly When arginine 383 was replaced by glycine, a small, neutral amino acid, the mutated protein was no longer expressed on the cell surface. Login to comment 256 ABCG2 p.Arg383Lys The R383K mutant, which preserved the strong positive charge at this position, was detectable on the cell surface and transported the tested substrates, though protein expression levels were significantly reduced. Login to comment 258 ABCG2 p.Arg383Ala Taken together, the results of the conservative lysine substitution and the in vitro translation experiments with the R383A mutant suggest that the role of arginine 383 is beyond that of simply anchoring the membrane. Login to comment 261 ABCG8 p.Arg405His It will be interesting to discover whether the corresponding R405H mutation in ABCG8 leads to similar effects on protein levels and trafficking as predicted by our data but not yet demonstrated in clinical samples, and if so, whether that mutant can be rescued in part or to any degree by pharmacological chaperones. Login to comment