ABCMdb

ABCG2 p.Arg383Gly

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Publications

PMID: 19406100 [PubMed] Polgar O et al: "Arginine 383 is a crucial residue in ABCG2 biogenesis."

No. Sentence Comment

3 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
41 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
80 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 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 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 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
106 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
110 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
113 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 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
137 Surface expression, function, protein and RNA levels of the R383A and R383G mutants transfected into HEK 293 cells. Login to comment
174 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 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
177 The expression level for the R383G mutant was too low to determine localization using confocal microscopy. Login to comment
178 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 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 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
195 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
206 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
253 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