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

17 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
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1788
Issue number7
DOIs
Publication statusPublished - Jul 1 2009

    Fingerprint

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

Polgar, O., Ediriwickrema, L. S., Robey, R. W., Sharma, A., Hegde, R. S., Li, Y., Xia, D., Ward, Y., Dean, M., Ozvegy-Laczka, C., Sarkadi, B., & Bates, S. E. (2009). Arginine 383 is a crucial residue in ABCG2 biogenesis. Biochimica et Biophysica Acta - Biomembranes, 1788(7), 1434-1443. https://doi.org/10.1016/j.bbamem.2009.04.016