ABCG2 p.Arg383Gly
| Predicted by SNAP2: | A: D (80%), C: D (80%), D: D (95%), E: D (91%), F: D (91%), G: D (91%), H: D (85%), I: D (85%), K: D (80%), L: D (85%), M: D (85%), N: D (85%), P: D (91%), Q: D (80%), S: D (85%), T: D (80%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Arginine 383 is a crucial residue in ABCG2 biogene... Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3. 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., [PMID:19406100]
Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. 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. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.
Comments [show]
None has been submitted yet.
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.
X
ABCG2 p.Arg383Gly 19406100:3:4
status: VERIFIED41 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].
X
ABCG2 p.Arg383Gly 19406100:41:40
status: VERIFIEDX
ABCG2 p.Arg383Gly 19406100:41:65
status: VERIFIED80 Generation of Sf9 cells expressing the R383A and R383G mutants Generation of transfer vectors containing wild-type ABCG2 has been described previously [30,31].
X
ABCG2 p.Arg383Gly 19406100:80:49
status: VERIFIED81 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.
X
ABCG2 p.Arg383Gly 19406100:81:44
status: VERIFIEDX
ABCG2 p.Arg383Gly 19406100:81:124
status: VERIFIED93 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].
X
ABCG2 p.Arg383Gly 19406100:93:67
status: VERIFIED100 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.
X
ABCG2 p.Arg383Gly 19406100:100:131
status: VERIFIED106 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).
X
ABCG2 p.Arg383Gly 19406100:106:43
status: VERIFIED110 The R383G mutant, as expected from its absence at the cell surface, did not display transport activity for either mitoxantrone or pheophorbide a.
X
ABCG2 p.Arg383Gly 19406100:110:4
status: VERIFIED113 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).
X
ABCG2 p.Arg383Gly 19406100:113:4
status: VERIFIED118 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).
X
ABCG2 p.Arg383Gly 19406100:118:228
status: VERIFIED137 Surface expression, function, protein and RNA levels of the R383A and R383G mutants transfected into HEK 293 cells.
X
ABCG2 p.Arg383Gly 19406100:137:70
status: VERIFIED174 Treatment with mitoxantrone resulted in a similar increase in the amount of the R383G mutant detected on the cell surface (data not shown).
X
ABCG2 p.Arg383Gly 19406100:174:80
status: VERIFIED175 To further analyze the localization of the R383A and R383G mutants in the mammalian cells, immunofluorescent staining followed by confocal microscopy was performed.
X
ABCG2 p.Arg383Gly 19406100:175:53
status: VERIFIED177 The expression level for the R383G mutant was too low to determine localization using confocal microscopy.
X
ABCG2 p.Arg383Gly 19406100:177:29
status: VERIFIED178 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].
X
ABCG2 p.Arg383Gly 19406100:178:23
status: VERIFIED180 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).
X
ABCG2 p.Arg383Gly 19406100:180:184
status: VERIFIED183 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.
X
ABCG2 p.Arg383Gly 19406100:183:280
status: VERIFIED195 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).
X
ABCG2 p.Arg383Gly 19406100:195:31
status: VERIFIED206 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.
X
ABCG2 p.Arg383Gly 19406100:206:24
status: VERIFIED253 When arginine 383 was replaced by glycine, a small, neutral amino acid, the mutated protein was no longer expressed on the cell surface.
X
ABCG2 p.Arg383Gly 19406100:253:5
status: VERIFIED