BREAST CANCER RESISTANCE PROTEIN - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 322

that eukaryotic ABC transporters require at least two nucleotide-binding domains
and two transmembrane domains for transport activity. 20 ABCG2 is considered a
half-transporter and is thought to homodimerize in order to function, as opposed to
other ABC half-transporters, which engage in heterodimeric association to form func-
tional transporters, a well-characterized example of which is the ABCG5/ABCG8
heterodimer. 21 ABCG2 homodimerization is supported by the fact that when ex-
pressed in Sf9 insect cells, where a heterodimeric partner is unlikely, the protein is fully
functional. Chimeric fusion proteins containing two ABCG2 monomers either with or
without a flexible linker peptide were also shown to be functional, endorsing the idea of
homodimer formation. 22 In addition, coimmunoprecipitation experiments using two
different tags on the ABCG2 monomers also suggested homodimer formation. 23 , 24
In general, little is known about the structure and dimerization of ABCG2. The most
accurate structural information could be obtained from high-resolution crystal struc-
tures; unfortunately, no human ABC transporter has been crystallized to date, and the
crystal structure of only a few full-length bacterial ABC transporters is available: the
lipid A transporter MsbA from Escherichia coli , 25 Vibrio cholera , 26 and Salmonella
typhimurium , 27 and the vitamin B 12 importer BtuCD from E. coli. 28 We should note,
however, that the MsbA structures were recently retracted.
The transmembrane domain of ABCG2, composed of residues 361 to 655, is pre-
dicted to have six transmembrane segments (TMs) and an extracellular loop between
transmembrane helices 5 and 6. This extracellular loop contains cysteine 603, which
has been demonstrated to form a symmetrical intermolecular disulfide bond in the
ABCG2 homodimer. 29 According to studies by multiple groups, this disulfide bridge
is not essential for transport and trafficking to the plasma membrane, given that the
C603A mutant displayed wild type-like characteristics. On the other hand, it has been
suggested that the other two cysteines of this extracellular loop, C592 and C608, form
intramolecular disulfide bonds, and that mutating these residues resulted in impaired
localization and function. 29
There are three putative N-linked glycosylation sites in the ABCG2 TMD (residues
418, 557, and 596), of which only asparagine 596 was shown to be glycosylated, and
this posttranslational modification seems not to be required for proper localization
and function of the transporter. 30 Asparagine 596 is located in the loop between TMs
5 and 6, helping to substantiate the prediction that this part of the protein is localized
extracellularly.
Sequence alignments with the previously mentioned bacterial ABC transporters of
known structure reveal a very low overall similarity for the ABCG2 TMD. This low
sequence similarity, together with the reversed orientation of ABCG2 (whose conse-
quences are not yet known), makes it difficult to speculate on the three-dimensional
structure of the ABCG2 TMD. According to a recently generated computer model, 31 a
cone-shaped large central cavity is formed when the 12 TMs, six from each monomer,
come together with twofold symmetry, similar to that seen in the MsbA homodimer.
Multiple points of interaction exist between the two monomers. One such interface
might be formed by the GXXXG motif in TM1 of ABCG2. This motif consists of two
glycines separated by any three amino acids and has been shown to be involved in the
dimerization of other membrane proteins, 32 the most well-characterized example of