Arginine 383 is a crucial residue in ABCG2 biogenesis

Orsolya Polgar, Lilangi S. Ediriwickrema, Robert W. Robey, Ajay Sharma, Ramanujan S. Hegde, Yongfu Li, Di Xia, Yvona Ward, Michael Dean, Csilla Ozvegy-Laczka, Balazs Sarkadi, Susan E. Bates

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1434-1443
Number of pages10
JournalBBA - Biomembranes
Volume1788
Issue number7
DOIs
Publication statusPublished - Jul 2009

Fingerprint

Mitoxantrone
Arginine
Substitution reactions
Glycosylation
Pharmacokinetics
Proteasome Endopeptidase Complex
Alanine
Glycine
Lysine
Adenosine Triphosphatases
Assays
Proteins
ATP-Binding Cassette Transporters
Adenosine Triphosphate
Cells
Degradation
Insects
Substrates
Cell Line
Mutation

Keywords

  • ABC transporter
  • ABCG2
  • Biogenesis
  • Membrane protein
  • Mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics
  • Medicine(all)

Cite this

Polgar, O., Ediriwickrema, L. S., Robey, R. W., Sharma, A., Hegde, R. S., Li, Y., ... Bates, S. E. (2009). Arginine 383 is a crucial residue in ABCG2 biogenesis. BBA - Biomembranes, 1788(7), 1434-1443. https://doi.org/10.1016/j.bbamem.2009.04.016

Arginine 383 is a crucial residue in ABCG2 biogenesis. / Polgar, Orsolya; Ediriwickrema, Lilangi S.; Robey, Robert W.; Sharma, Ajay; Hegde, Ramanujan S.; Li, Yongfu; Xia, Di; Ward, Yvona; Dean, Michael; Ozvegy-Laczka, Csilla; Sarkadi, Balazs; Bates, Susan E.

In: BBA - Biomembranes, Vol. 1788, No. 7, 07.2009, p. 1434-1443.

Research output: Contribution to journalArticle

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 2009, 'Arginine 383 is a crucial residue in ABCG2 biogenesis', BBA - Biomembranes, vol. 1788, no. 7, pp. 1434-1443. https://doi.org/10.1016/j.bbamem.2009.04.016
Polgar O, Ediriwickrema LS, Robey RW, Sharma A, Hegde RS, Li Y et al. Arginine 383 is a crucial residue in ABCG2 biogenesis. BBA - Biomembranes. 2009 Jul;1788(7):1434-1443. https://doi.org/10.1016/j.bbamem.2009.04.016
Polgar, Orsolya ; Ediriwickrema, Lilangi S. ; Robey, Robert W. ; Sharma, Ajay ; Hegde, Ramanujan S. ; Li, Yongfu ; Xia, Di ; Ward, Yvona ; Dean, Michael ; Ozvegy-Laczka, Csilla ; Sarkadi, Balazs ; Bates, Susan E. / Arginine 383 is a crucial residue in ABCG2 biogenesis. In: BBA - Biomembranes. 2009 ; Vol. 1788, No. 7. pp. 1434-1443.
@article{67e9e23d832a4f5493670f02b74f0b81,
title = "Arginine 383 is a crucial residue in ABCG2 biogenesis",
abstract = "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.",
keywords = "ABC transporter, ABCG2, Biogenesis, Membrane protein, Mutagenesis",
author = "Orsolya Polgar and Ediriwickrema, {Lilangi S.} and Robey, {Robert W.} and Ajay Sharma and Hegde, {Ramanujan S.} and Yongfu Li and Di Xia and Yvona Ward and Michael Dean and Csilla Ozvegy-Laczka and Balazs Sarkadi and Bates, {Susan E.}",
year = "2009",
month = "7",
doi = "10.1016/j.bbamem.2009.04.016",
language = "English",
volume = "1788",
pages = "1434--1443",
journal = "Biochimica et Biophysica Acta - Biomembranes",
issn = "0005-2736",
publisher = "Elsevier",
number = "7",

}

TY - JOUR

T1 - Arginine 383 is a crucial residue in ABCG2 biogenesis

AU - Polgar, Orsolya

AU - Ediriwickrema, Lilangi S.

AU - Robey, Robert W.

AU - Sharma, Ajay

AU - Hegde, Ramanujan S.

AU - Li, Yongfu

AU - Xia, Di

AU - Ward, Yvona

AU - Dean, Michael

AU - Ozvegy-Laczka, Csilla

AU - Sarkadi, Balazs

AU - Bates, Susan E.

PY - 2009/7

Y1 - 2009/7

N2 - 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.

AB - 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.

KW - ABC transporter

KW - ABCG2

KW - Biogenesis

KW - Membrane protein

KW - Mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=68149114480&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=68149114480&partnerID=8YFLogxK

U2 - 10.1016/j.bbamem.2009.04.016

DO - 10.1016/j.bbamem.2009.04.016

M3 - Article

VL - 1788

SP - 1434

EP - 1443

JO - Biochimica et Biophysica Acta - Biomembranes

JF - Biochimica et Biophysica Acta - Biomembranes

SN - 0005-2736

IS - 7

ER -