ABCMdb

ABCG2 p.Arg383Ala

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,

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[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.

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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
5 Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Login to comment
6 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 The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. 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
66 PCR-amplified wild-type ABCG2 or the R383A mutant from the appropriate pcDNA3.1 vectors were used as templates. 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
104 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 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 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
111 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
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
120 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 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 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 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 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 Surface expression, function, protein and RNA levels of the R383A and R383G mutants transfected into HEK 293 cells. Login to comment
143 (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 Proteasome/lysosome inhibition and "rescue" of the R383A mutant with mitoxantrone (MX). Login to comment
150 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 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 Localization of the R383A mutant in HEK 293 cells. Login to comment
166 The R383A mutant in Sf9 insect cells. Login to comment
169 (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 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 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
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
176 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
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
184 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
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
207 The R383A mutant was shown to be degraded by the proteasome via a kifunensine-insensitive pathway. Login to comment
228 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 The quality control pathway by which the R383A mutant is targeted to the proteasome seems to be kifunensine-insensitive. Login to comment
236 Interestingly, in the case of the R383A mutant, mitoxantrone seemed to help generate the mature, glycosylated protein. Login to comment
240 The R383A mutant localized to the cell surface and displayed some function in the HEK cells. Login to comment
241 When transfected to Sf9 insect cells, R383A was also detected on the surface, yet was unable to hydrolyze ATP. Login to comment
258 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

[hide] Posttranslational negative regulation of glycosyla... Biochem Biophys Res Commun. 2011 Jan 21;404(3):853-8. Epub 2010 Dec 22. Sugiyama T, Shuto T, Suzuki S, Sato T, Koga T, Suico MA, Kusuhara H, Sugiyama Y, Cyr DM, Kai H
Posttranslational negative regulation of glycosylated and non-glycosylated BCRP expression by Derlin-1.
Biochem Biophys Res Commun. 2011 Jan 21;404(3):853-8. Epub 2010 Dec 22., 2011-01-21 [PMID:21184741]

Abstract [show]
Human breast cancer resistance protein (BCRP)/MXR/ABCG2 is a well-recognized ABC half-transporter that is highly expressed at the apical membrane of many normal tissues and cancer cells. BCRP facilitates disposition of endogenous and exogenous harmful xenobiotics to protect cells/tissues from xenobiotic-induced toxicity. Despite the enormous impact of BCRP in the physiological and pathophysiological regulation of the transport of a wide variety of substrates, little is known about the factors that regulate posttranslational expression of BCRP. Here, we identified Derlin-1, a member of a family of proteins that bears homology to yeast Der1p, as a posttranslational regulator of BCRP expression. Overexpression of Derlin-1 suppressed ER to Golgi transport of wild-type (WT) BCRP that is known to be efficiently trafficked to the plasma membrane. On the other hand, protein expression of N596Q variant of BCRP, N-linked glycosylation-deficient mutant that preferentially undergoes ubiquitin-mediated ER-associated degradation (ERAD), was strongly suppressed by the overexpression of Derlin-1, whereas knockdown of Derlin-1 stabilized N596Q protein, suggesting a negative regulatory role of Derlin-1 for N596Q protein expression. Notably, knockdown of Derlin-1 also stabilized the expression of tunicamycin-induced deglycosylated WT BCRP protein, implying the importance of glycosylation state for the recognition of BCRP by Derlin-1. Thus, our data demonstrate that Derlin-1 is a negative regulator for both glycosylated and non-glycosylated BCRP expression and provide a novel posttranslational regulatory mechanism of BCRP by Derlin-1.

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No. Sentence Comment
163 Further, we could also assess the impact of Der- lin-1 on the expression of other BCRP variants such as R383A, G553L and G553E variants, which are also shown to be impaired N-linked glycosylation like N596Q BCRP [27,28]. Login to comment