ABCMdb

PMID: 19124053

McDevitt CA, Collins R, Kerr ID, Callaghan R
Purification and structural analyses of ABCG2.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):57-65. Epub 2008 Dec 13., 2009-01-31 [PubMed]

Sentences

No. Mutations Sentence Comment

717 ABCG2 p.Arg482Gly Perhaps the most significant variation in pharmacology arises with the R482G mutation, which dramatically alters the substrate specificity of ABCG2 and may provide clues into the site of drug interaction on theprotein [18,19]. Login to comment 722 ABCG2 p.Arg482Gly The substrate specificities for the wild-type and R482G isoforms have been reasonably well characterised with respect to drug transport and the ability to confer resistance [3,19]. Login to comment 725 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly Table 1 Investigations reporting ATPase activity of various ABCG2 isoforms Isoform ATPase activity Drug effect Reference Vmax Km (ATP) R482G (B) 45 nmol min-1 mg-1 - Prazosin IC50 =5 μM [61] Vi inhibits at 50 μM R482G (B) 15 nmol min-1 mg-1 - [21] (S) 32 nmol min-1 mg-1 WT (B) 27 nmol min-1 mg-1 (S) 29 nmol min-1 mg-1 WT (pure) (B) 357 nmol min-1 mg-1 2 mM Vi inhibits 60-80% [20] R482T (pure) (B) 1111 nmol min-1 mg-1 1 mM R482G (B) 10 nmol min-1 mg-1 Stimulated activity at 100 μM prazosin [57] (S) 30 nmol min-1 mg-1 WT (B) 40 nmol min-1 mg-1 - Activities are Vi sensitive [19] (S) 41 nmol min-1 mg-1 Activities reported at a fixed [ATP] and not full Michaelis-Menten analysis R482G (B) 65 nmol min-1 mg-1 (S) 140 nmol min-1 mg-1 R482T (B) 42 nmol min-1 mg-1 (S) 81 nmol min-1 mg-1 R482G (B) 70 nmol min-1 mg-1 - Prazosin IC50 =1 μM and fold-stimulation was 2X [30] (S) 150 nmol min-1 mg-1 The Michaelis-Menten characteristics for ATPase activity by a number of isoforms of ABCG2 are tabulated from a number of source references. Login to comment 732 ABCG2 p.Arg482Gly Wild-type and R482G ABCG2 isoforms are capable of ATP hydrolysis in the absence of any substrate although the Vmax for hydrolysis of 30-50 nmol Pi min-1 mg-1 is considerably lower than that shown for ABCB1. Login to comment 733 ABCG2 p.Arg482Gly The most pronounced stimulation of ATP hydrolysis by substrate has been demonstrated for the R482G mutant in the presence of micromolar concentrations of prazosin. Login to comment 734 ABCG2 p.Arg482Gly Unlike the R482G isoform, the wild-type protein was insensitive to stimulation by many drugs and increased nucleotide trapping in the presence of substrates could not be demonstrated [19]. Login to comment 745 ABCG2 p.Arg482Gly For example, mitoxantrone was more efficient at displacing [125 I]-IAA-Rh123 binding to wild-type versus R482G, but the latter isoform was able to confer greater levels of resistance to the anticancer drug. Login to comment 747 ABCG2 p.Arg482Gly An equilibrium and kinetic binding study on the R482G isoform of ABCG2 revealed the presence of multiple drug interaction sites on the protein [23]. Login to comment 760 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The first studies of ABCG2, and its pharmacologically differing isoforms (R482G, R482T), were conducted in drug selected mammalian cell lines due to the relative simplicity of inducing its expression by drug treatment [26,27]. Login to comment 768 ABCG2 p.Arg482Gly Janvilisri and co-workers utilised Lactococcus (L.) lactis as an expression system for wild-type and R482G ABCG2 [28,33] employing the nisin A-induced expression system that had previously been used for the expression of the prokaryotic ABC transporter LmrA [34]. Login to comment 795 ABCG2 p.Arg482Thr This study reported a percentage yield of ABCG2 of ~1.3% (membrane protein) and an approximately two fold lower yield for the R482T isoform. Login to comment 800 ABCG2 p.Arg482Gly Despite this lower affinity, a similar yield of ~1% was observed after IMAC for both the R482G isoform and the wild type (unpublished data). Login to comment 864 ABCG2 p.Arg482Gly A large oligomeric complex (molecular weight of ~600 kDa) of the R482G isoform was identified by Blue-native PAGE and electron microscopy [37]. Login to comment