Biomedical Engineering Reference
In-Depth Information
Subsequent to the cloning of ABCC1, additional members have been identified
that are all encoded by distinct genes and are often located on different chromosomes
(Table 11.1). The first evidence for the existence of more than one ABCC/MRP ef-
flux pump came from the cloning and sequencing of a novel 347-bp cDNA fragment
from normal rat liver, which was related to but not identical to rat
Abcc1
cDNA
and was not expressed in transport-deficient GY/TR¯ mutant rats.
33
These mutant
rats had long been known to lack the ATP-dependent biliary excretion of anionic
conjugates.
33
-
40
The 347-bp cDNA fragment was subsequently shown to be part of
the full-length cDNA encoding rat Abcc2/Mrp2,
41
which was identified as an ABCC1-
related protein located in the canalicular membrane of normal hepatocytes but not of
hepatocytes from transport-deficient mutant rats.
8
,
41
,
42
Because of this initial local-
ization in the hepatocyte canalicular membrane and its function as an efflux pump for
anionic conjugates, Abcc2 was formerly termed canalicular Mrp (cMrp)
41
or canalic-
ular multispecific organic anion transporter (cMoat).
42
The screening of expressed
sequence tag (EST) databases
26
,
27
,
43
,
44
or of the high-throughput genomic sequence
database
25
revealed the presence of additional ABCC/MRP efflux pumps and led
to the cloning and molecular characterization of the full-length cDNAs encoding
human ABCC3,
45
-
48
ABCC4,
22
,
49
-
53
ABCC5,
45
,
54
-
56
ABCC6,
24
,
57
,
58
ABCC10,
23
,
59
ABCC11,
25
-
27
,
60
and ABCC12
25
,
26
,
61
by several laboratories. The recently identified
ABCC13
gene encodes a truncated and likely functionally inactive protein.
62
,
63
All human
ABCC
genes have been analyzed with respect to their genomic organi-
zation (Table 11.1). Interestingly, four of the nine
ABCC/MRP
genes are located on
chromosome 16:
ABCC1
and
ABCC6
adjacent to each other in a tail-to-tail orientation
at region p13.1
24
and
ABCC11
and
ABCC12
adjacent to each other in a tail-to-head
orientation at region q12.1
25
,
26
indicating that each “gene tandem” originated by gene
duplication. A comparison of the genomic organization of human
ABCC1
,
ABCC2
,
and
ABCC3
showed remarkable similarities among all three genes with respect to
exon number and exon size; moreover,
ABCC1
,
ABCC2
, and
ABCC3
have 21 iden-
tical splice sites based on an amino acid alignment of the three respective proteins,
suggesting a close evolutionary relationship of these genes as well.
64
11.3. MOLECULAR CHARACTERIZATION
11.3.1. Domain Structure of the ABCC Proteins
Many studies have been performed to elucidate the structure of the ABCC proteins,
especially of ABCC1 and ABCC2. The ABCC transporters share with other members
of the ABC superfamily a typical core structure: the hydrophobic membrane-spanning
domains MSD1 and MSD2, each followed by a hydrophilic and highly conserved in-
tracellular nucleotide-binding domain: NBD1 and NBD2, respectively (Figure 11.1
a
).
Hydropathy profiles and limited proteolysis experiments suggested that ABCC1 con-
tains an additional hydrophobic amino terminal MSD0 of approximately 200 amino
acids, which precedes the typical core structure.
65
,
66
Glycosylation-site mutants and
epitope insertion revealed the presence of five additional transmembrane segments
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