ABCMdb

ABCG2 p.Arg383Lys

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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Publications
[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
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
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
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
185 Just as observed for the alanine mutant, R383K was represented by a double band on immunoblot suggestive of altered glycosylation. Login to comment
187 Surface expression, function and protein levels of the R383K mutant transfected into HEK 293 cells. Login to comment
188 (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 functionality of the R383K mutant was also assessed in flow cytometry-based transport assays. Login to comment
256 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