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FUNCTIONAL ANALYSIS OF AN α-HELICAL REGION IN THE HUMAN by
FUNCTIONAL ANALYSIS OF AN α-HELICAL REGION IN THE HUMAN
MULTIDRUG AND ORGANIC ANION TRANSPORTER MRP1
by
Steven V. Molinski
A thesis submitted to the Department of Pharmacology and Toxicology in conformity
with the requirements for the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
September, 2009
Copyright © Steven V. Molinski, 2009
ABSTRACT
Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa phosphoglycoprotein that
mediates the efflux of structurally diverse endo- and xenobiotics across biological
membranes, and is known to play roles in drug disposition and resistance. The goal of the
present study was to examine the functional importance of the region proximal to
transmembrane helix 17 (TM17) of MRP1 by mutational analysis of seven conserved
amino acids in this region. Thus, Glu1253, Glu1255, Val1261, Glu1262, Arg1263, Glu1266, and
Tyr1267 were initially replaced by Ala, and after expression in HEK293T cells, the
properties of the mutant proteins were investigated. All of the mutant proteins were
expressed at levels comparable to wild-type MRP1, indicating that these residues are not
critical for MRP1 biosynthesis. Vesicular transport assays showed that Ala-substitution
of Glu1253 and Glu1262 significantly reduced 17β-estradiol 17-(β-D-glucuronide) (E217βG)
and leukotriene C4 (LTC4) transport by 30-75% (p < 0.05), while Ala-substitution of
Glu1255 and Glu1266 had no effect. Transport activity of the same-charge mutant E1253D
was comparable to wild-type MRP1, while transport by E1262D remained reduced (by
50-75%) (p < 0.05). Kinetic analysis suggests that E1253A and E1262A exhibit reduced
E217βG uptake as a result of a decreased uptake affinity (Km), while the reduced transport
of E1262D was associated with a reduction in Vmax. Reciprocal mutations of potential
interhelical bonding partners of Glu1253 and Glu1262 (Lys1141 and Arg1142, respectively),
identified by examination of an atomic homology model of MRP1, did not significantly
enhance MRP1 function. This suggests that even if bonding interactions exist between
the side-chains of these two pairs of amino acids, the interactions are not exclusive. These
findings also suggest that Glu1253 and Glu1262 have unique and complex roles in substrate
ii
binding and/or translocation. Ala-substitution of Val1261, Arg1263 and Tyr1267 caused a
small reduction in E217βG transport (by 25-35%) (p < 0.05), while reductions in LTC4
transport were somewhat more substantial (by 30-55%) (p < 0.05). In conclusion, these
studies have provided the first evidence of the functional importance of anionic residues
in the COOH-proximal region of TM17 of MRP1.
iii
STATEMENT OF CO-AUTHORSHIP
This thesis is based on research conducted by Steven V. Molinski under the supervision
of Dr. Susan P.C. Cole. All data were obtained and analyzed by Steven V. Molinski.
Kathy Sparks provided assistance with tissue culture work. Dr. Gwenaëlle Conseil
provided assistance when optimizing the transfection efficiency of MRP1 expression
vectors, and generated K1141E and R1142E MRP1 mutant cDNA expression vectors as
described in Conseil et al. (2006).
iv
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my supervisor, Dr. Susan Cole, for giving me
the opportunity to work in her lab, as well as for allowing me to tailor my project to meet
my interests. I would also like to thank Susan for her guidance and support, as well as for
her excellent training which I believe has allowed me to become a better scientist. In
addition, I would like to thank the members of my advisory committee, Drs. Don
Maurice and Chris Nicol, for their helpful advice and insight.
I would also like to thank Drs. Gwenaëlle Conseil, Surtaj Iram, Tozammel Hoque,
and Kazuma Maeno for sharing their expertise, and also for their numerous helpful
discussions regarding various aspects of my project. I would also like to thank Kathy
Sparks for her assistance with cell culture work, as well as for sharing her experiences
with me. I must also thank Andrew, Marina, Xiaoqian, Fahad, and Leslie for their help
with research and non-research related subjects alike, as well as for their support during
the troubleshooting stages of my project. It was a pleasure to have worked alongside all
of you. Thank you for making my experience at Queen’s a memorable one.
Thank you to my family and friends for their support during my time in Kingston.
I would especially like to thank my parents and brother for their unconditional support
during my troubles and successes, as well as for their motivational talks during the last
stretch of my write-up.
Last but not least, I would like to thank Shujun Zhu for her support during these
past two years. You have always been there for me through the good times and bad, and
for this I am forever grateful.
v
TABLE OF CONTENTS
ABSTRACT........................................................................................................................ ii
STATEMENT OF CO-AUTHORSHIP ............................................................................ iv
ACKNOWLEDGEMENTS................................................................................................ v
TABLE OF CONTENTS................................................................................................... vi
LIST OF FIGURES ............................................................................................................ x
LIST OF TABLES............................................................................................................ xii
LIST OF ABBREVIATIONS.......................................................................................... xiii
CHAPTER I: INTRODUCTION AND LITERATURE REVIEW
1.1 ATP-Binding Cassette Trasporter Superfamily ............................................... 1
1.2 Multidrug Resistance ....................................................................................... 6
1.3 MRP1-Mediated Multidrug Resistance ........................................................... 7
1.4 Modulation of MRP1-Mediated Multidrug Resistance ................................. 10
1.5 Pharmacological, Toxicological, and Physiological Roles of MRP1 ............ 13
1.5.1 Pharmacological Roles of MRP1.................................................... 13
1.5.2 Toxicological Roles of MRP1 ........................................................ 14
1.5.3 Physiological Roles of MRP1......................................................... 16
1.6 Structure of MRP1 ......................................................................................... 19
1.7 MRP1 Transport Mechanism......................................................................... 22
1.8 Atomic Homology Models of MRP1............................................................. 24
1.9 MRP1 Substrate Specificity and Structure-Function Studies of MRP1 ........ 28
1.10 Rationale, Hypotheses, and Objectives........................................................ 30
vi
CHAPTER II: MATERIALS AND METHODS
2.1 Materials ........................................................................................................ 35
2.2 Secondary Structure Predictions .................................................................... 36
2.3 Sequence Alignments and In Silico Illustrations ........................................... 36
2.4 Vector Construction and Site-Directed Mutagenesis..................................... 36
2.5 Cell Culture.................................................................................................... 38
2.6 Transfections of MRP1 Expression Vectors in HEK293T Cells................... 39
2.7 Preparation of Membrane Vesicles from MRP1 Transfectants ..................... 39
2.8 Determination of MRP1 Protein Levels in Transfected Cells ....................... 40
2.9 MRP1-Mediated Transport of 3H-Labeled Substrates by Membrane
Vesicles .......................................................................................................... 41
2.10 Kinetic Analysis of [3H]E217βG Transport ................................................. 42
2.11 Photolabeling of MRP1 by [3H]LTC4 .......................................................... 43
2.12 Statistical Analyses ...................................................................................... 44
CHAPTER III: FUNCTIONAL ANALYSIS OF GLU1253, GLU1255, GLU1262 AND
GLU1266 MRP1 MUTANTS
3.1 Introduction..................................................................................................... 45
3.2 Results............................................................................................................. 47
3.2.1 Secondary Structure Predictions, Sequence Alignments, and In Silico
Illustrations of the Region COOH-Proximal to TM17 ...................... 47
3.2.2 Expression, and E217βG and LTC4 Transport Activities of AlaSubstituted MRP1 Mutant Proteins ................................................... 53
vii
3.2.3 Expression and Transport Activities of MRP1 mutants Same- and
Opposite-Charge Substitutions of Glu1253 and Glu1262....................... 56
3.2.4 Kinetic Analysis of [3H]E217βG Uptake by E1253A, E1262A, and
E1262DMRP1 Mutants...................................................................... 59
3.2.5 Photolabeling of E1253A, E1262A and E1262D MRP1 Mutants by
[3H]LTC4 ............................................................................................ 62
3.2.6 Expression, and E217βG and LTC4 Transport Activities of
K1141E/E1253K and R1142E/E1262R Double Reciprocal MRP1
Mutants .............................................................................................. 63
3.3 Discussion ....................................................................................................... 70
CHAPTER IV: FUNCTIONAL ANALYSIS OF ALA-SUBSTITUTED VAL1261,
ARG1263 AND TYR1267 MRP1 MUTANTS
4.1 Introduction..................................................................................................... 76
4.2 Results............................................................................................................. 77
4.2.1 Secondary Structure Predictions, Sequence Alignments, and In Silico
Illustrations of the Region COOH-Proximal to TM17 ...................... 77
4.2.2 Expression, and E217βG and LTC4 Transport Activities of AlaSubstituted MRP1 Mutant Proteins ................................................... 80
4.3 Discussion ....................................................................................................... 85
CHAPTER V: CONCLUSIONS AND FUTURE DIRECTIONS
5.1 Implications for MRP1 Structure.................................................................... 87
viii
5.2 Limitations of MRP1 Homology Models and Reciprocal Mutagenesis......... 90
5.3 Concluding Remarks....................................................................................... 91
REFERENCES ................................................................................................................. 94
APPENDIX
A1.1 Uncharacterized MRP1 Mutants E1079A, R1263K, and E1263E
Generated and Related to this Study......................................................... 112
A1.2 MRP1 Mutants R1202E, R1202E/E1204R, and R1202D/E1204K
Generated During the Course of this Master's Thesis in Collaboration with
Marina Chan.............................................................................................. 112
ix
LIST OF FIGURES
Figure 1.1 A predicted membrane topology model of MRP1 ........................................... 4
Figure 1.2 Phylogenetic tree of the human ABCC transporter subfamily......................... 5
Figure 1.3 Chemical structures of some MRP1 substrates ................................................ 9
Figure 1.4 An atomic homology model of MRP1 based on the crystal structure of
Sav1866 from S. aureus.................................................................................. 21
Figure 1.5 A hypothetical transport cycle of MRP1-mediated substrate efflux .............. 25
Figure 1.6 Helical projection of amino acids in the TM17-proximal region................... 32
Figure 3.1 Secondary structure and in silico illustration of the TM17-proximal region of
MRP1 .............................................................................................................. 46
Figure 3.2 Structural predictions and sequence alignments of the TM17-proximal region
of MRP1.......................................................................................................... 48
Figure 3.3 Putative relative locations and side-chain positions of Glu1253, Glu1255, Glu1262
and Glu1266 in a homology model of MRP1.................................................... 50
Figure 3.4 In silico illustration of potential paired interhelical interactions involving sidechains of Glu1253 and Lys1141, Glu1255 and Arg1138, Glu1262 and Arg1142, and
Glu1266 and Arg1075 .......................................................................................... 52
Figure 3.5 Expression levels and vesicular uptake of 3H-labeled organic anions by
Ala-substituted Glu1253, Glu1255, Glu1262 and Glu1266 MRP1 mutants............ 54
Figure 3.6 Expression levels and vesicular uptake of 3H-labeled organic anions by sameand opposite-charge substitutions of Glu1253 and Glu1262 MRP1 mutants ..... 57
x
Figure 3.7 Kinetic analysis of [3H]E217βG uptake by Ala-substituted Glu1253 and Glu1262,
and Asp-substituted Glu1262 MRP1 mutants ................................................... 60
Figure 3.8 Photolabeling of Ala-substituted Glu1253 and Glu1262, and Aspsubstituted Glu1262 MRP1 mutant proteins by [3H]LTC4................................ 64
Figure 3.9 Expression levels and vesicular uptake of E217βG and LTC4 by MRP1
mutants containing single and double exchange mutations of Lys1141 and
Glu1253 ............................................................................................................ 65
Figure 3.10 Expression levels and vesicular uptake of E217βG and LTC4 by MRP1
mutants containing single and double exchange mutations of Arg1142 and
Glu1262 .......................................................................................................... 67
Figure 4.1 Structural predictions and sequence alignments of Val1261, Arg1263, and
Tyr1267 of MRP1.............................................................................................. 78
Figure 4.2 Putative relative locations and side-chain positions of Val1261, Arg1263, and
Tyr1267 in a homology model of MRP1........................................................... 79
Figure 4.3 In silico illustration of potential interhelical interactions involving sidechains of Val1261 and Val1083, Arg1263 and Glu1079, and Tyr1267 and Phe1063 ... 81
Figure 4.4 Expression levels and vesicular uptake of E217βG and LTC4 by Alasubstituted Val1261, Arg1263, and Tyr1267 MRP1 mutants................................ 83
xi
LIST OF TABLES
Table 1.1 Genetic disorders associated with mutant ABC transporters............................. 2
Table 1.2 Reported tissue distribution of MRP1 mRNA and protein.............................. 15
Table 1.3 Selected substrates of MRP1 and reported Km values ..................................... 17
Table 3.1 Estimated distances between side-chains of Glu1253, Glu1255, Glu1262 and
Glu1266, and other amino acids which potentially form interhelical bonds in
MRP1 ............................................................................................................... 51
Table 3.2 Summary of kinetic parameters of [3H]E217βG uptake by Ala-substituted
Glu1253 and Glu1262, and Asp-substituted Glu1262 MRP1 mutants.................... 61
Table 4.1 Estimated distances between the side-chains of Val1261, Arg1263 and Tyr1267,
and other amino acids in other helices which might form bonding interactions
........................................................................................................................................... 82
xii
LIST OF ABBREVIATIONS
ABC
ATP-binding cassette
ALD
Adrenoleukodystrophy
BSEP
Bile salt export pump
CFTR
Cystic fibrosis transmembrane conductance regulator
E13SO4
Estrone 3-sulfate
E217βG
17β-estradiol 17-(β-D-glucuronide)
GSH
Glutathione
HEK
Human embryonic kidney
Kir
Inward rectifying potassium channel
LTC4
Leukotriene C4
MAb
Monoclonal antibody
MDR
Multidrug resistance
MRP
Multidrug resistance protein
MSD
Membrane spanning domain
NBD
Nucleotide binding domain
NBS
Nucleotide binding site
P-gp
P-glycoprotein
TM
Transmembrane
TSB
Tris-sucrose buffer
xiii
CHAPTER I: INTRODUCTION AND LITERATURE REVIEW
1.1 ATP-Binding Cassette Transporter Superfamily
ATP-binding cassette (ABC) transporters are polytopic membrane proteins that are found
in all species, and require ATP to translocate structurally diverse endo- and xenobiotic
compounds across the lipid bilayer of cell membranes (Higgins, 1992). Mammalian ABC
transporters are involved in many different physiological processes, including: 1) cellular
efflux of signaling and bioactive molecules (e.g. eicosanoids, bile acids, and steroid
conjugates), 2) modulating absorption, distribution, and elimination of nutrients and their
metabolites, and 3) preventing accumulation, and aiding elimination of drugs and
chemical toxins and their metabolites from a variety of tissues in the human body
(Szakács et al., 2008). When certain ABC transporters are expressed in human tumour
cells, the latter function can also lead to a multidrug resistance (MDR) phenotype
(Gerlach et al., 1987; Gottesman et al., 2002; Leonard et al., 2003). Therefore, these
proteins are of great physiological, pharmacological, and pathological importance.
In addition to regulating the disposition of endogenous and exogenous organic
molecules and conferring resistance to multiple chemotherapeutics, mutations in certain
human ABC transporters are responsible for a number of genetic disorders (Table 1.1).
Among these are Harlequin-type ichthyosis (ABCA12), Dubin-Johnson Syndrome
(MRP2/ABCC2), Pseudoxanthoma elasticum (MRP6/ABCC6), and cystic fibrosis
(CFTR/ABCC7) (Akiyama, 2006; Kartenbeck et al., 1996; Ringpfeil et al., 2000;
Riordan et al., 1989).
1
Gene
Symbol
Protein
(common
name)
Genetic Disorder
References
ABCA1
ABCA3
Tangier disease
Newborn respiratory distress syndrome
ABCA4
ABCA12
ABCB2
ABCB3
ABCB4
Stargardt disease
Harlequin-type ichthyosis
Ankylosing spondylitis
Ankylosing spondylitis
Progressive familial intrahepatic
cholestasis type 3
X-linked sideroblastosis and anemia
Progressive familial intrahepatic
cholestasis type 2
Dubin-Johnson Syndrome
Dean, 2005
Klugbauer and
Hofmann, 1996
Dean, 2005
Akiyama et al., 2006
Feng et al., 2009
Feng et al., 2009
Dean, 2005
TAP1
TAP2
MDR2
ABCB7
ABCB11
BSEP
ABCC2
MRP2
ABCC6
ABCC7
ABCC8
MRP6
CFTR
SUR1
ABCC9
SUR2
ABCC11
ABCD1
ABCG5
MRP8
ALD
ABCG8
Pseudoxanthoma elasticum
Cystic fibrosis
Familial persistent hyperinsulinemic
hypoglycemia of infancy
Dilated cardiomyopathy with ventricular
tachycardia
Dry ear wax, body odour
Adrenoleukodystrophy
Sitosterolemia
Sitosterolemia
Dean, 2005
Dean, 2005
Kartenbeck, et al.,
1996
Ringpfeil et al., 2000
Riordan et al., 1989
Dean, 2005
Dean, 2005
Yoshiura et al., 2006
Dean, 2005
Rudkowska and
Jones, 2008
Rudkowska and
Jones, 2008
Table 1.1: Genetic disorders associated with mutant ABC transporters.
2
The human ABC transporter superfamily is comprised of 49 genes that are
distributed among 7 phylogenetic branches designated A through G (Dean and Allikmets,
2001). The basic functional structure of ABC proteins consists of two membrane
spanning domains (MSDs) and two nucleotide binding domains (NBDs) (Higgins, 1992),
and some members contain an additional MSD, the function of which is not yet clear
(Bakos et al., 1998; Westlake et al., 2005). The domains of functional ABC transporters
are usually arranged as MSD-NBD-MSD-NBD, while the third MSD can be found at the
N-terminus of 7 members of the ABC‘C’ subfamily (i.e. ABCC1, ABCC2, ABCC3,
ABCC6, ABCC8, ABCC9, and ABCC10) (Figure 1.1) (Higgins, 1992; Deeley et al.,
2006). Many mammalian ABC proteins are translated as one long polypeptide, while
some are translated into two polypeptides each consisting of either an NBD-MSD (e.g.
ABCG2 (breast cancer resistance protein, BCRP)) or an MSD-NBD (e.g. TAP1/TAP2
(transporter associated with antigen processing 1 and 2)), and thus these 2-domain
‘subunits’ dimerize to form a functional 4-domain transporter.
The human ABC‘C’ subfamily consists of 13 members, including the multidrug
resistance proteins MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), MRP4
(ABCC4), MRP5 (ABCC5), MRP6 (ABCC6), MRP7 (ABCC10), MRP8 (ABCC11),
MRP9 (ABCC12) and the pseudogene MRP10 (ABCC13), as well as CFTR (ABCC7),
and the sulfonylurea receptors SUR1 (ABCC8) and SUR2 (ABCC9) (Figure 1.2) (Dean
and Allikmets, 2001). CFTR is a cAMP-gated chloride channel, and SUR1 and SUR2 are
regulators of inwardly rectifying potassium channels (Kir) (Riordan et al., 1989; Bryan et
al., 2007), while the nine MRPs appear to be mainly involved in transporting organic
anions out of cells, as is discussed below (Deeley et al., 2006). MRP1 (ABCC1) is
3
MSD0
TM
H2N
1
2
3 4
MSD1
5
6
7
8
MSD2
9 10 11
12 13 14 15 16 17
Y
Y
Y
Lipid
Bilayer
COOH
NBD2
NBD1
Figure 1.1: A predicted membrane topology model of MRP1.
A topology model of MRP1 showing MSD0, MSD1, MSD2, NBD1, and NBD2 coloured
in light gray, green, blue, yellow, and magenta, respectively. TM17 is coloured in red,
and the orange star denotes the TM17-proximal region investigated in this thesis. The ‘Yshaped’ sticks represent N-glycosylation sites. MSD, membrane spanning domain; TM,
transmembrane α-helix; NBD, nucleotide binding domain.
4
ABCC1 (MRP1)
ABCC3 (MRP3)
ABCC2 (MRP2)
ABCC6 (MRP6)
ABCC4 (MRP4)
ABCC7 (CFTR)
ABCC5 (MRP5)
ABCC11 (MRP8)
ABCC12 (MRP9)
ABCC10 (MRP7)
ABCC8 (SUR1)
ABCC9 (SUR2)
Figure 1.2: Phylogenetic tree of the human ABCC transporter subfamily.
Alignments were performed using ClustalW v2.0 software
(http://www.ebi.ac.uk/Tools/clustalw2/index.html). Shown in parentheses are the
common protein names for each ABCC transporter. The pseudogene ABCC13 has been
omitted from this analysis.
5
the MRP family member most strongly implicated in mediating MDR in malignant
disease (Abe et al., 1994; Borst, 1999; Berger et al., 2005; Haber et al., 2006; Ozben et
al., 2006).
1.2 Multidrug Resistance
MDR is a major obstacle to overcome during cancer chemotherapy since the appearance
of this phenotype is typically associated with treatment failure and a poor prognosis
(Gerlach et al., 1986; Gottesman et al., 2002). Cells which exhibit MDR are able to
survive exposure to normally lethal doses of multiple cytotoxic agents. Thus, MDR to a
broad range of structurally and functionally diverse agents might explain why
chemotherapeutic regimens that use 'cocktails' of multiple anticancer drugs with different
targets are not always more effective than single agent treatments (Gottesman et al.,
2002).
There are two major forms of MDR, inherent and acquired. Inherent MDR refers
to the ability of a cell to exhibit relative resistance without having to modify its own
intracellular processes. These processes prevent adequate doses of active anticancer drugs
from reaching their target site (Luqmani, 2005; McCarthy, 2009). Acquired MDR is a
multi-factorial process, allowing for tumours, which are initially sensitive to cytotoxic
agents, to develop resistance to a spectrum of unrelated drugs through multiple
mechanisms and pathways. Thus, acquired MDR refers to the ability of a cell to adapt to
its local environment by modifying its genetic expression profile (i.e. upregulating genes
that promote survival and/or downregulating genes that do not), such that the amount of
6
drug that reaches its target is reduced, or the apoptotic and other pathways that are set
into motion by the drug/target interaction are blunted (Bradley et al., 1988).
MRP1 together with P-glycoprotein (P-gp) (ABCB1) and ABCG2 are major
players in intrinsic and acquired MDR in many tumours (Borst, 1999; Pankunlu et al.,
2003; Leonard et al., 2003; Burger et al., 2005; Pajic et al., 2009). There has been much
research aimed at modifying drug efflux mediated by these transporters in human cancers
as one approach to combat this deadly phenotype.
1.3 MRP1-Mediated Multidrug Resistance
MRP1 was discovered in 1992 during investigations into the cause of MDR in a human
small cell lung carcinoma cell line, designated H69AR (Mirski et al., 1987; Cole et al.,
1992). An unusual characteristic of this cell line was that it did not overexpress P-gp, the
only protein demonstrated to cause MDR at that time (Mirski et al., 1987; Cole et al.,
1991; Cole et al., 1992). An mRNA transcript, encoded by a gene later designated
ABCC1, was found to be markedly overexpressed, and was predicted to encode a protein
containing 1531 amino acids with a minimum molecular mass of 170 kDa (Cole et al.,
1992). ABCC1 was localized to chromosome 16p13.11-p13.12, and determined to contain
31 exons that span 192.84 kb (Cole et al., 1992; Grant et al., 1997; Dean and Allikmets,
2001). The 6.5 kb mRNA transcript encoded by ABCC1 was translated into a ~190 kDa
phosphoglycoprotein when expressed in mammalian cells (Cole et al., 1994; Almquist et
al., 1995). In addition, MRP1 was shown to confer resistance to, and mediate the ATPdependent efflux of a broad range of structurally diverse anticancer compounds,
7
including anthracyclines, Vinca alkaloids, antifolates, and an epipodophyllotoxin in
cultured cells (Figure 1.3) (Cole et al., 1994; Deeley et al., 2006).
Subsequent studies aimed at determining the prevalence of MRP1 in human
tumours were able to show a correlation of MRP1 protein expression with the stage of
disease, providing some evidence for a role for MRP1 in clinical MDR (Filipits et al.,
1999; Haber et al., 2006; Ohsawa et al., 2005; Larbcharoensub et al., 2008; Hendig et al.,
2009). Thus, MRP1 is expressed at relatively high levels in certain tumours, including
those derived from lung, colorectal, testis, and breast tissues (Berger et al., 2005; Meijer
et al., 1999; Bart et al., 2004; Filipits et al., 1999).
Several studies have investigated whether MRP1 expression can be used as a
prognostic indicator for determining the effectiveness of cancer chemotherapies. For
example, Filipits et al. (2007) collected tumour samples from ~800 patients with
completely resected non-small cell lung cancer, and by immunohistochemical analysis
with the MRP1-specific antibody MRPr1, showed that although MRP1 was frequently
expressed, it was not a good predictive tool for determining clinical outcomes in
cisplatin-based adjuvant chemotherapy. This is not necessarily unexpected, since there is
no evidence that MRP1 confers resistance to cisplatin (Sharp et al., 1998). Another
immunohistochemical study of 115 ovarian carcinoma patients also concluded that
response to chemotherapy, as well as prognoses did not correlate well with MRP1
expression levels. Again, however, this study involved samples from patients receiving
platinum-based chemotherapy (Arts et al., 1999).
In contrast to the above two studies, MRP1 protein expression was found to be a
good predictive indicator of poor clinical outcome in several smaller studies of patients
8
A
Methotrexate
Etoposide
Vincristine
Doxorubicin
B
Leukotriene C4
Estrone 3-sulfate
17β-estradiol 17-(β-D-glucuronide)
Figure 1.3: Chemical structures of some MRP1 substrates.
A, anticancer agents; B, established, and potential physiological substrates.
9
with nasopharyngeal carcinoma (Larbcharoensub et al., 2008), primary neuroblastoma
(Haber et al., 2006), nodal diffuse large B-cell lymphoma (Ohsawa et al., 2005), breast
carcinoma (Filipits et al., 1999), and retinoblastoma (Hendig et al., 2009). Interestingly,
in the study by Larbcharoensub et al. (2008), cisplatin-based chemotherapy was
employed, and thus MDR could not be attributed to MRP1 alone. Nevertheless, these
authors concluded that MRP1 protein expression could be used as a prognostic marker in
certain tumour tissue samples, while noting that there are currently no guidelines or
standardized testing for MRP1 expression in clinical studies. Additionally, other proteins
involved in MDR, such as P-gp and ABCG2, could also be used as clinical prognostic
markers, but again there are currently no standardized guidelines for this approach,
although recommendations have been made for P-gp (Orina et al., 2009).
Positive correlations in certain tumour tissues provide a potential target for
improving response to cancer chemotherapy, in that the suppression of MRP1 expression
or inactivation of its drug efflux activity might be expected to enhance the
chemosensitivity of cells in the patient’s tumour(s). However, it should be kept in mind
that other ABC efflux transporters, including P-gp and ABCG2, can also be
overexpressed in conjunction with MRP1, making it even more difficult to correlate
disease state solely with MRP1-based MDR, and thus further complicating the course of
treatment (Huang and Sadée, 2005).
1.4 Modulation of MRP1-Mediated Multidrug Resistance
Antisense oligonucleotides can be used to target MRP1 mRNA in order to reduce MRP1
protein levels so that, in theory, the response to chemotherapy can be improved. The use
10
of antisense oligonucleotides has been shown to reduce the effects of MRP1 in vitro. For
example, Stewart et al. (1996) used an MRP1 antisense phosphorothioate oligonucleotide
targeted to the coding region to reduce MRP1 mRNA levels in lung cancer cells by 90%,
as well as MRP1 protein by 50%. Similarly, Canitrot et al. (1996) found that a modified
2’-deoxy analog of the same antisense oligonucleotide greatly reduced (70%) MRP1
protein levels in transfected HeLa cells. In addition, Niewiarowski et al. (2000)
demonstrated that MRP1 mRNA and protein levels can be significantly reduced in
leukemia cells (75% and 50%, respectively), using antisense oligonucleotides targeted to
different regions of the MRP1 mRNA, and Kuss et al. (2002) showed that MRP1 protein
levels can be significantly reduced (by 60%) in an in vivo mouse-human xenograft model
of neuroblastoma. More recently, Matsumoto et al. (2004) showed that antisense
oligonucleotides, again targeting different sequences, significantly reduced MRP1 mRNA
levels (by 85%) in an etoposide resistant glioma cell line, and Pakunlu et al. (2004)
demonstrated that antisense oligonucleotides significantly reduced (by 35%) MRP1
protein levels in the H69AR small cell lung cancer cell line. Although proof of concept
has been demonstrated by these in vitro and in vivo studies, suppression of MRP1 will not
be as effective for multifactorial (i.e. transporter and non-transporter-mediated) clinical
MDR. Furthermore, this approach may not be feasible due to poor delivery at target sites.
A variety of small molecules of both natural and synthetic origin have also been
investigated as MRP1 inhibitors primarily in vitro. Compounds such as LTD4 receptor
(CysLT1 and CysLT2) antagonists (e.g. MK571 and BAY u9773), polyhydroxylated
sterols (e.g. acetate agosterol A), dietary bioflavonoids, tricyclic isoxazole derivatives
(e.g. LY475776), benzothiophene derivatives (e.g. LY329146), as well as
11
pyrazolopyrimidines (e.g. CBLC4H10 (Reversan)) have been shown to inhibit MRP1mediated multidrug resistance by interacting with the transporter. The IC50 values for
many of these compounds are in the low μM range, while others are considerably more
potent (e.g. LY475776 is in the low nM range, although it is dependent on GSH for its
activity) (Mao et al., 2002; Boumendjel et al., 2005; Burkhart et al., 2009; Maeno et al.,
2009).
Agosterol A and its analogs have been shown to competitively inhibit MRP1mediated LTC4 transport in a GSH-dependent manner in vitro, while in vivo results have
not yet been reported (Ren et al., 2003). Similarly, both LY475776 and LY329146 were
also able to competitively inhibit MRP1-mediated LTC4 transport in a GSH-dependent
manner in vitro, while LY171883 and BAY u9773 were able to competitively inhibit
MRP1-mediated E217βG transport, further suggesting an inhibitory role for these small
molecules (Mao et al., 2002; Norman et al., 1999; Maeno et al., 2009).
Dietary bioflavonoids are never specific inhibitors of MRP1 (or other ABC
proteins, and other enzymes), and often inhibit the activity of MRP2 and P-gp, as well as
other intracellular proteins (e.g. topoisomerase II, an enzyme crucial for chromosome
segregation) (Di Pietro et al., 2002; Morris and Zhang, 2006). In addition, the inhibitory
action of some bioflavonoids is enhanced in the presence of GSH (Leslie et al., 2003a). It
has been suggested that bioflavonoids might be used in conjunction with other
modulatory agents in order to achieve synergistic inhibition of MRP1 (Boumendjel et al.,
2005). The inhibitory effects of MK571 are also not specific for MRP1, as it is reported
to modulate the activity of many MRPs, including MRP1, MRP2, MRP3, and MRP4 with
varying potencies (Gekeler et al., 1995; Chen et al., 1999). Such relatively non-specific
12
inhibitors are likely to be of little clinical value since they are likely to alter other
physiologically relevant processes in addition to MRP1-mediated MDR (Bakos et al.,
2000).
The newly reported pyrazolopyrimidine Reversan was able to inhibit MRP1mediated export of vincristine and etoposide, in murine models of neuroblastoma
(syngeneic and human xenografts) with no apparent off-target toxicities (Burkhart et al.,
2009). These data support the idea that small molecules targeting MRP1 could potentially
be used to combat tumours exhibiting MRP1-mediated MDR by increasing their
sensitivity to conventional cytotoxic chemotherapeutic agents.
1.5 Pharmacological, Toxicological, and Physiological Roles of MRP1
As mentioned previously, in addition to conferring resistance in malignant cells, MRP1
also plays important roles in drug and toxin disposition in normal cells (Leslie et al.,
2005; Szakács et al., 2008). Thus, some of the main roles of MRP1 include tissue defense
from toxic compounds, as well as mediating the passage of endo- and xenobiotics
through cellular and tissue barriers, and these are discussed briefly below.
1.5.1 Pharmacological Roles of MRP1
MRP1 protein is found on the basolateral membrane of polarized epithelial and
endothelial cells in a wide range of tissues, as well as the apical membrane of specialized
endothelial cells in the blood-brain barrier (reviewed by Deeley et al., 2006). MRP1
mRNA and protein is present at relatively low levels in all tissues throughout the body,
and relatively higher levels are found in the lung, testis, skeletal and cardiac muscles,
13
kidney, prostate, and the placenta (Table 1.2) (Deeley et al., 2006; Wijnholds et al., 1997;
Cole et al., 1992; Kruh et al., 1995; Szakács et al., 2008). This tissue distribution pattern
of MRP1 is consistent with its role in regulating the accumulation of pharmacological
agents at a number of blood-organ interfaces, like the blood-cerebral spinal fluid (CSF)barrier and the blood-testis barrier, that create so-called pharmacological sanctuary sites
in the body (Leslie et al., 2005). To date, in vivo studies have been very useful in
determining the pharmacological consequences of deficient MRP1 protein expression.
Studies involving Mrp1 knockout mice have shown that certain tissues (e.g. lung, testis)
exhibit an increased sensitivity to acute doses of some chemotherapeutic agents,
including etoposide and vincristine, although sensitivity to other agents, including
cisplatin and sodium arsenite, was not changed (Wijnholds et al., 1997; Lorico et al.,
1996; Rappa et al., 1999; Johnson et al., 2001; van Tellingen et al., 2003).
1.5.2 Toxicological Roles of MRP1
MRP1 is also important in the cellular efflux of a number of toxins including the
glucuronide conjugate of the hydroxylated metabolite (NNAL) of 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK), a carcinogen found in cigarette smoke (Leslie et al.,
2001a). NNK is first metabolized to NNAL and after glucuronidation, the NNAL-O-Gluc
conjugate can be exported by MRP1 across the plasma membrane. However, despite
being an organic anion, NNAL-O-Gluc requires the presence of GSH (or a non-reducing
analog, e.g. S-Me-GSH) to be transported, for reasons that are still not well understood
(Leslie et al., 2001a). Interestingly, MRP2 also transports NNAL-O-Gluc but without a
14
Tissue
MRP1 mRNA
MRP1 Protein
Blood-brain barrier
Brain
Breast
Colon
Heart
Kidney
Liver
Lung
Marrow
Ovary
Pancreas
Placenta
Prostate
Skeletal muscle
Skin
Small intestine
Spleen
Testis
++
++
+
++
+
+++
+
+++
+
+
+
+++
+
+++
+
++
+
+++
++
+
+
++
+
++
(very low)
+++
+
+
+
++
+
++
++
++
+
+++
Table 1.2: Reported tissue distribution of MRP1 mRNA and protein.
‘+’, ‘++’, and ‘+++’ denote low, moderate, and high levels of expression, respectively.
Data were obtained from reviews by Leslie et al., 2001b; Leslie et al., 2005; Deeley et al.,
2006; Ballatori et al., 2009. These values represent whole tissue analysis, and thus do not
reflect cell-type specific expression within these tissues. It should be noted that
variability among studies as well as inconsistencies in the literature has been reported.
15
requirement for GSH. Furthermore, MRP1 is able to transport the GSH conjugate of the
liver and lung carcinogen (and mycotoxin) aflatoxin B1 (Zaman et al., 1995).
In addition, MRP1 also mediates the transport of many unconjugated toxins. For
example, MRP1 has been shown to protect cells from sodium arsenite, sodium arsenate,
and potassium antimony tartrate (Cole et al., 1994; Loe et al., 1997; Rappa et al., 1997).
It is also interesting to note that certain exogenous chemicals are reported to
induce MRP1 protein expression. For example, the hepatotoxin carbon tetrachloride has
been shown to dose-dependently increase Mrp1 protein levels in mice liver, while tertbutyl hydroquinone, and quercertin have been shown to induce MRP1 protein expression
in a human breast cancer cell line (Aleksunes et al., 2006; Yamane et al., 1998; Schrenk
et al., 2001). Although several chemicals appear to alter the expression patterns of MRP1,
the mechanism(s) by which they do so is not fully understood.
1.5.3 Physiological Roles of MRP1
It has been well established that MRP1 transports GSH, glucuronide, and sulfate
conjugates of certain endobiotics, even though there are no apparent common structural
features of these moieties, other than their organic anion nature (Figure 1.3) (Loe et al.,
1996a). Well characterized endogenously formed substrates of human MRP1 include
17β-estradiol 17-(β-D-glucuronide) (E217βG), estrone 3-sulfate (E13SO4), and LTC4
(Table 1.3) (Deeley et al., 2006; Cole and Deeley, 2006; Leier et al., 1994). Despite their
different chemical structures, E217βG and LTC4 are competitive inhibitors of each other’s
transport by MRP1 (Jedlitschky et al., 1996; Loe et al., 1996a). However, many sitedirected mutagenesis studies have identified regions of MRP1 which exhibit substrate
16
Substrates
Apparent Km (μM)
References
Endogenous metabolites
Bilirubin*
Monoglucuronosyl bilirubin
Bisglucuronosyl bilirubin
DHEAS*
E13SO4
E13SO4*
E217βG
0.01
N/D
N/D
5.0
4.2
0.7
1.5; 2.5
1.0
1.6
Rigato et al., 2004
Jedlitschky et al., 1997
Jedlitschky et al., 1997
Zelcer et al., 2003
Qian et al., 2001
Qian et al., 2001
Jedlitschky et al., 1996;
Loe et al., 1996a
Salerno et al., 2001
Leslie et al., 2003a
Loe et al., 2000
Leier et al., 1996
Jedlitschky et al., 1994;
Loe et al., 1996b
Evers et al., 1997
Renes et al., 2000
3.6
0.2
N/D
2150
290
39
28
Jedlitschky et al., 1996
Loe et al., 1997
Xiao et al., 2005
Zeng et al., 2001
Leslie et al., 2001b
Leslie et al., 2001a
Zaman et al., 1996
GSH
GSH**
GSH***
GSSG
LTC4
PGA2-SG
4-Hydroxynonenal-SG
Exogenous compounds
2,4-Dinitrophenyl-SG
Aflatoxin B1-SG
Depsipeptide FK228
Methotrexate
Metolachlor-SG
NNAL-O-glucuronide*
Ethacrynic acid-SG
3400
116
83
93
0.1
Table 1.3: Selected substrates of MRP1 and reported Km values.
‘*’ denotes Km determined in the presence of GSH; ‘**’ denotes Km determined in the
presence of apigenin. ‘***’ denotes Km determined in the presence of verapamil. N/D, not
determined. Km values were determined by in vitro vesicular transport assays in all cases.
17
selectivity, in that certain mutations can affect transport of LTC4 but not E217βG, and
vice-versa, suggesting these organic anions bind to distinct yet overlapping binding sites
(Haimeur et al., 2002; Ito et al., 2001a). However, the potencies of several non-GSHcontaining modulators including MK571, BAY u9773, and LY171883 are not affected by
either type of mutation (Maeno et al., 2009). Taken together, these studies suggest that
MRP1 has at least three substrate/modulator binding sites: one that interacts with E217βG,
one that interacts with LTC4, and one that interacts with neither E217βG nor LTC4.
Another piece of evidence which supports the hypothesis that MRP1 has multiple binding
sites is that NNAL-O-glucuronide can inhibit both LTC4 and GSH transport, but not
E217βG transport, suggesting that the chemical structure of the parent compound (and not
the glucuronide moiety) determine where the metabolite binds (Leslie et al., 2001b;
Zelcer et al., 2003).
MRP1 also mediates the transport both GSH and GSSG, although the affinity
(Km) for GSSG is reportedly ~40 times higher than for GSH (Salerno et al., 2001; Leier et
al., 1996; Heijn et al., 1997). Lorico et al. (1997) generated Mrp1 knockout mice and
found that GSH levels were increased (by 25-90%) in tissues known to express Mrp1
compared to wild-type mice. Similarly, Rappa et al. (1997) generated an Mrp1 knockout
embryonic stem cell line and found that GSH levels were increased compared to wildtype cells, in this case two-fold. Efflux of both GSH and GSSG is relevant, since it
implies a role for MRP1 in controlling the intracellular GSH:GSSG ratio, an indicator of
oxidative stress (Cole and Deeley, 2006; Ballatori et al., 2009). Thus, MRP1 may efflux
GSSG in order to maintain an optimal GSH:GSSG ratio, so that oxidative stress can be
controlled during cytotoxic conditions (e.g. during the intracellular accumulation of
18
reactive metabolites) (Mueller et al., 2005). In addition, it has been shown that MRP1mediated transport of vincristine, aflatoxin B1, and daunorubicin can be enhanced in the
presence of GSH (Loe et al., 1996b; Loe et al., 1997; Renes et al., 1999). Inhibition of
MRP1 by certain modulators, including several bioflavonoids (e.g. apigenin), agosterol A,
and several tricyclic isoxazole derivatives (e.g. LY475776), has also been shown to be
dependent on, or stimulated by, GSH (Loe et al., 2000; Leslie et al., 2003a; Ren et al.,
2001; Mao et al., 2002). Furthermore, MRP1-mediated efflux of GSH potentiates
oxidative stress levels in H69AR-drug selected cells, which can then lead to tumour cell
death by induction of apoptosis (Laberge et al., 2007).
Additionally, MRP1 can protect against the accumulation of endogenous toxins.
Thus, MRP1 can mediate the removal of unconjugated bilirubin from the brain (Rigato et
al., 2004). This is important because high bilirubin levels are toxic to many tissues, and
prolonged exposure (e.g. in newborns with jaundice) can cause irreversible damage
(Chang et al., 2009). Furthermore, although Mrp1 knockout mice are viable and fertile,
they display an impaired inflammatory response, which likely is associated with defective
transport of LTC4 from leukotriene-synthesizing mast cells (Wijnholds et al., 1997).
These in vivo studies corroborate earlier in vitro studies reporting the importance of
MRP1 in mediating LTC4 transport (Leier et al., 1994; Loe et al., 1996).
1.6 Structure of MRP1
Based on both biochemical evidence and protein folding algorithms, it has been
determined that MRP1 is likely comprised of seventeen transmembrane (TM) α-helices
arranged into three MSDs (MSD0, MSD1 and MSD2), and two intracellular NBDs
19
(Figure 1.1) (Bakos et al., 1996; Hipfner et al., 1997). MSD0 contains five TM helices
(TMs 1-5), while MSD1 and MSD2 each contain six TM helices, TMs 6-11 and 12-17,
respectively (Hipfner et al., 1997). As mentioned previously, MRP1 is considered a
‘long’ ABCC transporter (along with MRP2, 3, 6, and 7), due to the presence of MSD0 at
the NH2-proximal end of these transporters (Bakos et al., 1998; Borst et al., 2000).
Current evidence strongly supports a model where MSD1 and MSD2 provide the
translocation pathway for substrates, and the two NBDs form a ‘sandwich’ dimer capable
of binding and hydrolyzing ATP (Figure 1.4) (Higgins et al., 2004).
As mentioned earlier, the function of MSD0 is not entirely understood. Firstly,
this domain (usually defined as amino acids 1-203) does not appear to directly participate
in substrate translocation, since its deletion up to amino acid 203 does not substantially
affect LTC4 transport (Bakos et al., 1998). Some evidence suggests that the linker region
(cytoplasmic loop 3, CL3; residues 208-270) joining MSD0 to MSD1 contains a
redundant trafficking signal, such that when removed, the MSD1-NBD1/MSD2-NBD2
‘core’ is still trafficked to the plasma membrane correctly and retains transport activity,
but when both the CL3 and the COOH-proximal trafficking signals (located within the
COOH-terminal 30 residues) are removed, MRP1 is not expressed on the plasma
membrane (Westlake et al., 2005; Deeley et al., 2006). However, mutations of individual
amino acids in this region do adversely affect the transport of LTC4 (Deeley et al., 2006).
Post-translational modifications of MRP1 include glycosylation and
phosphorylation. MRP1 is glycosylated at Asn19, Asn23, and Asn1006, and this information
was useful in establishing that the NH2 terminus as well as the loop connecting TM12
20
out
in
Figure 1.4: An atomic homology model of MRP1 based on the crystal structure of
Sav1866 from S. aureus.
The homology model of MRP1 shown was generated using the crystal structure of
Sav1866 from S. aureus as template (DeGorter et al., 2008). MSD1, MSD2, NBD1, and
NBD2 are coloured green, blue, yellow, and magenta, respectively. TM17 and the TM17proximal region are coloured red and orange, respectively. The thick horizontal lines
represent the putative location of the lipid bilayer. Note that MSD0 is absent from this
homology model. When rotated 90o and viewed from the extracellular side, a putative
translocation pathway can be observed. Images were generated using PyMOL v1.1.
21
and TM13 are extracellular (Figure 1.1) (Hipfner et al., 1997). In contrast, the extent and
sites of phosphorylation are currently unknown, although preliminary evidence suggests
that Ser and Thr, but not Tyr residues are phosphorylated (Ma et al., 1995; Almquist et
al., 1995; Almquist, Loe, and Cole, unpublished). A recent inspection of the MRP1
protein sequence using current phosphorylation site prediction software (NetPhos 2.0,
http://www.cbs.dtu.dk/services/NetPhos; KinasePhos 2.0,
http://kinasephos2.mbc.nctu.edu.tw) indicates the presence of 40 possible serine
phosphorylation sites, and 7 threonine sites.
Although electron microscopy structural data of MRP1 are of low resolution (~22
Å), they indicate that MRP1 contains a “pore” in the centre of the molecule (Rosenberg et
al., 2001). Additionally, based on crystal structures of both the bacterial ABC transporter
Sav1866 and murine P-gp, the core structure of MRP1 (i.e. MSD1-NBD1/MSD2-NBD2)
is believed to be comprised of TM helices that are substantially twisted and somewhat
flexible, allowing for each MSD to interact with the same and opposite NBD (DeGorter
et al., 2008; Dawson and Locher, 2006; Aller et al., 2009). Twisting of the TMs is
thought to promote communication between MSD1 and NBD1/2, as well as between
MSD2 and NBD1/2, and thus allowing for substrate(s) to be appropriately accommodated
during the various conformations adopted by MRP1 throughout the active transport
process (Dawson and Locher, 2006; DeGorter et al., 2008).
1.7 MRP1 Transport Mechanism
The ATPase activity of ABC transporters including MRP1 requires three highly
conserved motifs found within each NBD – the Walker A, Walker B, and ‘C’ active
22
transport signature motifs. The Walker A and Walker B motifs of NBD1, in cooperation
with the ‘C’ motif of NBD2, form a composite nucleotide binding site (NBS) which acts
as a pocket to bind and hydrolyze ATP, as well as release ADP/Pi (Dawson and Locher,
2006). Similarly, the Walker A and Walker B motifs of NBD2 interact with the ‘C’
signature motif from NBD1 to form a second NBS.
The NBDs of ABCC proteins, including MRP1, are unlike most other ABC
transporters, since they are functionally and structurally non-equivalent (Deeley et al.,
2006). In MRP1 (and presumably other MRPs which share similar distinctive structural
features), NBD1 has a higher affinity for ATP than NBD2, while NBD2 has a higher
hydrolytic activity (Gao et al., 2000; Hou et al., 2000). The ATPase activity of purified
MRP1 is ~100-fold lower than that of some ABC transporters which have functionally
equivalent NBDs (e.g. prokaryotic ABC transporters and mammalian P-gp) and the nonequivalency of the NBDs of MRP1 may contribute to this (Mao et al., 1999; Mao et al.,
2000).
Using azido-derivatized nucleotides as photoactive cross-linking agents, it has
been demonstrated that ATP binding by NBD1 of MRP1 can occur independently of
NBD2 in vitro, while the binding of ATP by NBD2 appears dependent on the binding of
ATP to NBD1 (Gao et al., 2000; Qin et al., 2008). A significant structural difference
between NBD1 and NBD2 is the absence of 13 amino acids between the Walker A and
‘C’ signature motifs in NBD1 that are present in NBD1 of P-gp (Cole et al., 1992; Hung
et al., 1998; Yuan et al., 2001). In addition, the higher hydrolytic activity of NBD2 has
been attributed to the presence of a Glu residue immediately following the Walker B
motif, which enhances the cleavage of the high energy β-γ phosphodiester bond of ATP,
23
while an Asp residue at this same position in NBD1 is believed to be responsible for the
enhanced ATP binding (Qin et al., 2008). The latter has been suggested due to the fact
that mutation of Glu to Asp in NBD2 significantly enhanced azido-ATP binding and
decreased azido-ATP hydrolysis (Payen et al., 2003).
The current models of the transport cycle of MRP1 have many similarities
regarding the steps which must occur for substrate translocation, but the order in which
these events take place has not yet been established (Deeley et al., 2006; Rothnie et al.,
2006). In one simplified model, substrate first binds to a high-affinity site(s) on the
cytoplasmic side of MRP1 in its inward facing conformation, which in turn induces
conformational changes that enhances the binding of ATP by NBS1, as it facilitates the
formation of a ‘sandwich’ dimer with both NBDs (Figure 1.5) (Deeley et al., 2006;
Szakács et al., 2008). The binding of a second molecule of ATP by NBS2 is facilitated
after binding of ATP by NBS1, which alters the conformation of the NBD ‘sandwich’
dimer, thus signaling to the MSDs to ‘open’ the transporter ‘outward’ and decrease its
affinity for its substrate. The release of ADP/Pi from one or both of the NBSs induces
changes in the structure of MRP1, resulting in the resetting of the transporter to its
original high-affinity conformation so that it may undergo another cycle of transport
(Chang, 2007).
1.8 Atomic Homology Models of MRP1
X-ray crystallography, electron microscopy, and nuclear magnetic resonance imaging are
currently the most common methods used to determine the structure of proteins, but these
methods remain limited in their ability to elucidate the structure of large hydrophobic
24
3-5
1
2
out
in
+ Pi
Substrate
2 ATP( )
+ Pi
2 ADP( ) + 2 Pi
Figure 1.5: A hypothetical transport cycle of MRP1-mediated substrate efflux.
In this model of substrate translocation: 1-2, substrate is recognized by MRP1 and binds
to an intracellular site, which in turn induces conformational changes that enhance the
binding of ATP by NBS1 and NBS2; 3-5, ATP binding and hydrolysis induce changes in
the conformation of the NBD ‘sandwich’ dimer, and these changes are transmitted to the
MSDs causing the transporter to ‘open’ and the affinity of MRP1 for substrate to
decrease (substrate is subsequently released into the extracellular space), while release of
ADP/Pi from one or both of the NBSs resets MRP1 back to its original high-affinity
conformation so that it may undergo another cycle of transport. Modified from Deeley et
al. (2006), DeGorter et al. (2008), and Aller et al. (2009).
25
mammalian proteins (Chayen and Saridakis, 2008; Rosenberg et al., 2001; Hollenstein et
al., 2007). Thus, solved structures of membrane proteins currently represent <1% of all
structures available in the Research Collaboratory for Structural Bioinformatics’ Protein
Data Bank (Hurwitz et al., 2006; http://www.rcsb.org/pdb/home/home.do).
As mentioned before, existing electron microscopy structural data of MRP1 are of
low resolution, and thus are unable to provide information on the precise arrangement of
the TM helices and NBDs in the functional protein (Rosenberg et al., 2001; Higgins et al.,
2004). Crystallization of mammalian MRP1 (and other human ABC proteins) poses a
significant challenge, mainly due to their large size and polytopic nature, as well as the
difficulty of isolating large amounts of purified active protein (Wu et al., 2005; Chayen
and Saridakis, 2008; Dawson and Locher, 2006). For this reason, structural information
on MRP1 and other human ABCs is primarily inferred from models based on solved
structures of bacterial ABC transporters (Dawson and Locher, 2006).
The first atomic homology models of human ABC transporters were based on the
crystal structures of the E. coli and V. cholera lipid A transporter MsbA (Chang et al.,
2001; Chang, 2003a). MsbA is a homodimeric transporter with each monomer consisting
of one MSD (containing six TMs), as well as one NBD (Chang, 2003a). In an initial
study, two MsbA monomers constituting the active transporter were aligned to MSD1NBD1 and MSD2-NBD2 of MRP1, and the 4-domain core structure of MRP1 was
modeled using computational software (Campbell et al., 2004). However, in 2006 it
became evident that the structures of the E. coli and V. cholera MsbA did not agree with
the structure of another homologous bacterial ABC transporter from S. aureus, Sav1866
(Dawson and Locher, 2006). Errors in the structures of E. coli and V. cholera MsbA were
26
due in part to mistaken interpretations of the handedness, caused by inversions in the
diffraction data, which affected the topology of both structures (Chang, 2003b). In light
of these mistakes, the MsbA structural data were retracted, and the validity of MsbAbased models of human ABCs including MRP1 discredited (Miller, 2006). Structures of
the homodimeric ABC transporter from S. aureus, Sav1866, in both ADP- and ATPbound states have been solved at high resolutions (3.0 and 3.4 Å, respectively) (Dawson
and Locher, 2006; 2007). Subsequently, the crystal structure of Sav1866 was used as a
template to develop new atomic homology models of mammalian ABC proteins
including MRP1 (DeGorter et al., 2008) and P-gp (Zolnerciks et al., 2007; O’Mara and
Tieleman, 2007).
Recently, a crystal structure of nucleotide-free mouse P-gp has been reported to a
resolution of 3.8 Å (Aller et al., 2009). In this study, P-gp was crystallized into identical
forms in the presence and absence of two recently identified inhibitors, QZ59-SSS and
QZ59-RRR (Aller et al., 2009). This is unexpected because it might have been
anticipated that P-gp would be in different conformations when an inhibitor is bound or
absent. Another unexpected observation was the estimated distance between the NBDs,
which at ~30 Å represents a substantial amount of space for the NBDs to move through
to come together to form a ‘sandwich’ dimer as required during the transport process.
This large distance is energetically unfavorable and not easily explained. Indeed, a recent
review commenting on the study by Aller et al. (2009) suggests that this form of P-gp is
unlikely to be physiologically relevant (Gottesman et al., 2009). Nevertheless, the
discovery of crystallization conditions by this study at least provides important new
27
information that may be useful to those investigating crystal structures of other ABC
transporters, including MRP1.
In the meantime, as mentioned, the structure of Sav1866 has been used as a
template to model the core 4-domain structure of MRP1 and other ABCs (e.g. P-gp,
MRP4, MRP5) (O’Mara and Tieleman, 2007; El-Sheikh et al., 2008; Ravna et al., 2007;
DeGorter et al., 2008). Each monomer of Sav1866 has moderate sequence similarity to
MRP1, in that Sav1866 is approximately ~22% identical to each ‘half’ (MSD1-NBD1
and MSD2-NBD2) of MRP1, although most of this is in the NBDs (DeGorter et al.,
2008). These Sav1866-based models of MRP1 were derived so that they could be used to
guide the design and interpretation of biochemical studies (DeGorter et al., 2008).
However, it should be remembered that these models represent only a ‘snapshot’ of just
one (i.e. low-affinity state; nucleotide-bound, substrate-free) of the many conformations
assumed by MRP1 (and other ABCCs) during the complex transport process, and thus
models the positions of amino acids in only a single conformation.
1.9 MRP1 Substrate Specificity and Structure-Function Studies of MRP1
As described above in Sections 1.3 and 1.5, MRP1 mediates the transport of a wide range
of structurally diverse conjugated and unconjugated organic anions, and drugs and toxins.
Other MRPs share the ability to transport some of these organic anions, with MRP2
having a substrate transport profile most similar to MRP1, although the affinity of the
two homologs for a given organic anion can differ substantially (Cole and Deeley, 2006).
Extensive biochemical analyses have been very useful in identifying specific domains
and amino acids that are important for the structure and function of these transporters.
28
Using site-directed mutagenesis, a large number of mutation-sensitive residues
have been identified that are critical for overall transport activity, substrate specificity,
and/or stable expression of MRP1 in the plasma membrane (Deeley and Cole, 2006). In
addition, this approach has uncovered specific regions of MRP1 which are critical for
helix packing (using ‘reciprocal’ or ‘double exchange’ mutagenesis) (Haimeur et al.,
unpublished), inter-domain communication (Koike et al., 2004; Conseil et al., 2009), and
ATPase activity (Letourneau et al., 2007; Qin et al., 2008).
Some mutagenesis studies have been based on testing the assumption that amino
acids conserved among MRP1 and its orthologs are likely to be functionally important.
They have also targeted specific residues that have distinctive biophysical properties (e.g.
polarity, ionizability, aromaticity, helix breaking) (Deeley and Cole 2006). For example,
it has been shown that mutation of certain ionizable residues in TM6 (i.e. Lys332, His335)
causes substrate selective changes, such that LTC4 transport is eliminated or reduced
while E217βG transport remains intact (Haimeur et al., 2002). In contrast, mutation of
certain polar or aromatic residues in TM17 (i.e. Thr1242, Tyr1243, Asn1245, Trp1246) mainly
reduces just E217βG transport, and not LTC4 (Ito et al., 2001; Zhang et al., 2001, 2002).
Consistent with the importance of residues in TM6 and TM17 for substrate specificity,
Bao et al. (2005) reported that LTC4 transport was eliminated by poly-Ala substitution of
TM6, whereas LTC4 transport was not affected by poly-Ala substitution of TM17.
Unfortunately, this study did not determine the transport of substrates other than LTC4,
and so it is not clear how the TM helix substitution will affect the transport of other
substrates by MRP1.
29
Using a combination of photolabeling and mass spectrometry techniques, Wu et
al. (2005) identified regions of MRP1 that interact with LTC4. Thus, after photolabeling
purified MRP1 with LTC4, MALDI-TOF mass spectrometry identified short sequences in
TM6, TM7, TM10, TM17, and part of CL3 as regions of modification by LTC4 (Wu et
al., 2005). These results are somewhat inconsistent with previous site-directed
mutagenesis studies, in that, for example, residues in TM17 have been shown to be less
important for LTC4 transport (Ito et al., 2001a; Zhang et al., 2001; Zhang et al., 2002).
Further studies involving tandem mass spectrometry (i.e. MS/MS) are needed to identify
the specific residues that interact with LTC4, and whether they differ at the different
stages of the transport cycle of MRP1.
1.10 Rationale, Hypotheses, and Objectives
As described above, previous studies of MRP1 have demonstrated that many charged,
aromatic, and polar residues within the TMs of MSD1 and MSD2 are mutation sensitive,
and thus may contribute directly or indirectly to one or more of the substrate binding sites,
proper folding of the protein, and/or the ATPase activity of this transporter. Therefore,
amino acids in the core structure of MRP1 play multiple roles in structure and function.
The COOH-terminal TM of MRP1, TM17 (which we have defined here as
residues 1228-1248), is very amphipathic (Ito et al., 2001). Substrate selective transport
and/or binding activities become apparent when certain TM17 residues with aromatic or
polar properties are replaced (Ito et al., 2001a; 2001b; Zhang et al., 2001, 2002; Situ et
al., 2004; Ren et al., 2002). On the other hand, both LTC4 and E217βG transport were
eliminated when the TM17-proximal residue Arg1249 was mutated (including replacement
30
with the same charge amino acid Lys) (Ren et al., 2002; Situ et al., 2004). This adjacent
Arg1249 may affect overall activity by affecting structure rather than just a substrate
binding site. Thus, Arg1249 may assist in anchoring this TM within the plasma membrane
by interacting with the negatively charged head groups of the phospholipids in the inner
leaflet of the bilayer, and possibly by forming a cation-π bond with nearby Trp1246. Thus,
the role of residues in the intracellular α-helical extension of TM17 may differ from
residues within TM17 itself, in that residues in TM17 may be part of a substrate binding
site, while the TM17-proximal region may be more involved in overall MRP1 structure
and thus overall function.
Accordingly, we first hypothesized that the helical extension of TM17 (residues
1249-1269), which is predicted to be located in the cytoplasm where it continues into
NBD2, is important for MRP1 structure and function. To test this hypothesis, four highly
conserved acidic residues (Glu1253, Glu1255, Glu1262, and Glu1266) and three other highly
conserved residues (Val1261, Arg1263, and Tyr1267) in this region of MRP1 were chosen for
investigation by site-directed mutagenesis (Figures 1.1 and 1.6).
Current homology models suggest that the TM17-proximal region of MRP1 may
be in close proximity to the cytoplasmic helical extensions from TM14 and TM15,
raising the possibility of interhelical bonding interactions between the side-chains of
these helices with those of the TM17-proximal region. Consequently, we also
hypothesized that such interhelical side-chain interactions may play a role in the stable
expression and/or function of MRP1. To test this hypothesis, reciprocal (exchange)
mutations of selected amino acids in the TM17-proximal region with amino acids in the
cytoplasmic extensions of TM14 or TM15 were generated and functionally characterized.
31
1249
RMSSEMETNIVAVERLKEYSE
1269
Y
1267
T
R
1256
R
1249
A
1260
1263
1253
E
L
S 1252
1264
1257 N
V 1259
E
1250 M
1266
E
1268 S
1261
1255
1262
1251
E
V
1254
S
1258
I
1265
M
K
1269
E
Figure 1.6: Helical projection of amino acids in the TM17-proximal region.
The TM17-proximal region (residues 1249-1269) is shown as a linear sequence and as a
helical projection. The helical projection, or helical ‘wheel’, begins at Arg1249 and
continues as a right-handed α-helix ‘down’ the z-axis ‘into’ the page. Non-polar, polar
uncharged, acidic, and basic residues are coloured orange, green, pink, and blue,
respectively. This image was generated using the Helical Wheel Applet
(http://cti.itc.virginia.edu /~cmg/Demo/wheel/wheelApp.html) and Adobe Photoshop
v5.0.
32
Thus, to test the hypotheses that (i) conserved residues found in the TM17-proximal
region of MRP1 are important for its expression and/or function, and (ii) at least some of
these residues participate in bonding interactions with the side-chains of residues in the
cytoplasmic extensions of TM14 and/or TM15, the following objectives were pursued:
1. Glu residues at positions 1253, 1255, 1262 and 1266, as well as Val1261, Arg1263
and Tyr1267, were replaced with Ala, and the effects on MRP1 expression in
HEK293T cells and transport activity in inside-out membrane vesicles were
determined.
2. For those Glu mutants exhibiting an altered phenotype, same charge and opposite
charge mutations were also created to determine whether the charge or other
characteristics (e.g side-chain volume) of the Glu residue were important for
MRP1 function.
3. Based on in silico analyses of a homology model of MRP1 which identified
potential interhelical bonding partners of the above seven conserved residues,
single and double reciprocal mutants of two pairs of amino acids, Glu1253 and
Lys1141, and Glu1262 and Arg1142, were created, and MRP1 expression and
transport activity determined as before.
In this thesis, the materials and methods used for all of the studies listed in the
objectives above are described in Chapter II. The results obtained from the
characterization of the four Glu mutants, and the Glu1253/Lys1141 and Glu1262/Arg1142
double exchange mutants are presented and discussed in Chapter III, while the results
obtained from the characterization of the Val1261, Arg1263 and Tyr1267 single mutants are
33
presented and discussed in Chapter IV. Finally, Chapter V includes an overall discussion
of the results described in Chapters III and IV, and proposes future directions of research.
34
CHAPTER II: MATERIALS AND METHODS
2.1 Materials
[14,15,19,20-3H(n)]LTC4 (160 Ci mmol-1), [6,7-3H(n)]E217βG (45 Ci mmol-1), and
Western Lightning Plus-Enhanced Chemiluminescence (ECL) blotting substrate were
from Perkin Elmer Life Sciences (Woodbridge, ON). E217βG, AMP, ATP, benzamidine,
bovine serum albumin (BSA), Dulbecco’s Modified Eagle’s Medium (DMEM),
GenEluteTM Plasmid Miniprep kit, Kodak X-Omat LS film for autoradiography, and 2mercaptoethanol were from Sigma-Aldrich (Oakville, ON). Creatine kinase, creatine
phosphate, and protease inhibitors (EDTA-free) were from Roche (Mississauga, ON).
LTC4 was purchased from Calbiochem (La Jolla, CA) and AmplifyTM was from GE
Healthcare (formerly Amersham; Milwaukee, WI). Film for immunoblots was purchased
from Ultident Inc. (St. Laurent, QC) and Bradford protein assay kit was from Bio-Rad
Laboratories (Hercules, CA). Fetal bovine serum was purchased from Gibco-Invitrogen
(Grand Island, NY). Lipofectamine 2000TM transfection reagent was from Invitrogen
(Burlington, ON). Mouse anti-human MRP1 MAb QCRL-1 was previously generated by
Hipfner et al. (1994) and its epitope determined as MRP1 amino acids 918-924 (Hipfner
et al., 1996). Rabbit anti-Na+/K+ ATPase antibody (sc-28800) was purchased from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated goat antimouse and goat anti-rabbit IgG antibodies were from Pierce Biotechnology (Rockford,
IL). PageRuler Plus protein ladder was from Fermentas International Inc. (Burlington,
ON) and polyvinylidene fluoride (PVDF) membranes were from Pall Corporation (East
Hills, NY).
35
2.2 Secondary Structure Predictions
Algorithms used for secondary structure predictions of MRP1 included DSC, MLRC,
PHD, and Predator (default parameters, performed May 2008), and can be found at
http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html.
2.3 Sequence Alignments and In Silico Illustrations
ClustalW 1.0 (default parameters, performed May 2008; http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html) was used to perform sequence
alignments. PyMOL 1.1 (DeLano Scientific, Palo Alto, CA; http://www.pymol.org) was
used to view the homology model of MRP1 (DeGorter et al., 2008), and to estimate
distances between the side-chains of amino acids.
2.4 Vector Construction and Site-directed Mutagenesis
The template for mutagenesis was prepared by cloning a 2.0 kb XmaI fragment from
pcDNA3.1(–)-MRP1K (containing nucleotides 2337-4322 of the MRP1 sequence
encoding amino acids 780-1440) into pGEM3Z (Promega, Madison, WI) (Ito et al.,
2001a). Mutations were generated using the following sense primers, with wild-type
MRP1 pGEM3Z plasmid (nucleotides 2337-4322) as template (Integrated DNA
Technologies Inc., Coralville, IA) (substituted nucleotides are underlined, and silent
mutations used to introduce restriction sites to facilitate characterization are italicized):
E1253A, 5’-CGG ATG TCA TCT GCC ATG GAA ACC-3’; E1253D, 5’-CGG ATG
TCA TCT GAC ATG GAA ACC-3’; E1253K, 5’-CGG ATG TCA TCT AAA ATG
GAA ACC-3’; E1255A, 5’-CTG GAA ATG GCC ACC AAC ATC GTG-3’; V1261A,
36
5’-C ATC GTG GCC GCG GAG AGG CTC-3’; E1262A, 5’-C GTG GCC GTG GCC
AGG CTC AAG G-3’; E1262D, 5’-GTG GCC GTG GAT AGG CTC AAG GAG-3’;
E(D)1262E, 5’-GTG GCC GTG GAG AGG CTC AAG GAG-3’; E1262R, 5’- GTG
GCC GTG AGA AGG CTC AAG GAG-3’; R1263A, 5’-G GCC GTG GAA GCG CTC
AAG GAG-3’; R1263K, 5’-GTG GCC GTG GAG AAG CTC AAG GAG-3’; R1263E,
5’-GTG GCC GTG GAG GAA CTC AAG GAG-3’; E1266A, 5’-GAG GCT CAA GGC
ATA TTC AGA GAC-3’; Y1267A, 5’-GG CTC AAG GAA GCT TCA GAG ACT
GAG-3’. Generation of MRP1 mutants K1141E and R1142E has been previously
described (Conseil et al., 2006). Sense primers included: K1141E, 5’-CGG CAG CTG
GAG CGC CTG GAG TCG GTG AGC-3’; R1142E, 5’-CCC GGC AGC TGA AGG
AGC TCG AGT CGG TCA GCC G-3’. K1141E/E1253K and R1142E/E1262R double
mutant constructs were generated by site-directed mutagenesis using E1253K and
E1262R sense/antisense primers, with K1141E and R1142E mutant constructs as
template, respectively.
Mutations were generated using polymerase chain reaction (PCR). PCR was
performed in a PTC-100 Programmable Thermo Controller (MJ Research Inc.,
Watertown, MA), and for each substitution the following components were used: 1 µl
(100 ng) of MRP1 pGEM3Z plasmid DNA (nucleotides 2337-4322) as template, 1.25 µl
(125 ng) of both sense and anitsense mutant DNA primers, 5 µl of 10X PfuTurbo
reaction buffer (Stratagene, La Jolla, CA), 4 µl of 2.5 mM dNTPs, 1 µl (2.5U) of
PfuTurbo DNA polymerase (Stratagene), and 36.5 µl of Milli-Q water (total reaction
volume of 50 µl). PCR was carried out under the following conditions: step 1, 94 oC for
45 sec; step 2, (primer Tmelt)-5 oC for 1 min, step 3, 72 oC for 10 min; step 4, repeat steps
37
1-3 for 18 cycles. Reactions were stopped by incubating tubes at 4 oC, and then 1 µl
(10U) of DpnI was added to each reaction tube for 60 min at 37 oC to remove methylated
parental template DNA. XL1-Blue competent (E. coli) cells (50 µl) were transformed
with MRP1 mutant constructs (40 µl PCR volume) by first using a 30 min incubation at 4
o
C, followed by a 90 sec ‘heat shock’ at 42 oC. Transformed cells were then incubated at
4 oC for 2 min, shaken at 37 oC for 30 min, plated on LB-Ampicillin selective agar plates,
and then incubated at 37 oC overnight. Colonies were picked after 16-18 h, and plasmid
DNA was prepared using the GenEluteTM Plasmid Miniprep kit (Sigma).
The presence of Ala substitutions in the pGEM3Z plasmids was initially
confirmed by appropriate digests using NcoI, MscI, SacII, MscI, Eco47III, and HindIII
for E1253A, E1255A, V1261A, E1262A, R1263A and Y1267A mutant constructs,
respectively. The presence of mutation(s) in pGEM3Z constructs, including those already
determined by diagnostic digest, were further confirmed by DNA sequencing (TCAG
Inc., Toronto, ON). A 1.5 kb BsmBI/ClaI-fragment containing each mutation(s) in
pGEM3Z was subcloned back into the pcDNA3.1(–)-MRP1K expression vector, and this
1.5 kb fragment was again sequenced to confirm the integrity of the cloned region.
2.5 Cell Culture
HEK293T cells (ATCC CRL-11268) were maintained in DMEM supplemented with 4
mM L-glutamine and 7.5% FBS in 175-cm2 flasks by Kathy Sparks. All cultures were
grown at 37 oC in 5% CO2/ 95% air in a Series II water-jacketed CO2 incubator with
HEPA filtration (Thermo Scientific, Waltham, MA).
38
2.6 Transfection of Human Embryonic Kidney Cells
Transient transfections were performed as previously described, with small modifications
to cell seeding density and the transfection reagent used (Ito et al., 2001a). Briefly, wildtype pcDNA3.1(–)-MRP1K and mutant MRP1 expression vectors were transfected into
HEK293T cells that had been seeded at 20 x 106 cells per 20 ml in 150 mm plates.
Twenty-four h later (at 80-90% confluence), MRP1 cDNA expression vectors (20 μg in
40 µl, 260/280 nm ratio ~1.8), purified using GenEluteTM Plasmid Miniprep kits (Sigma),
were incubated with 75 µl Lipofectamine 2000TM reagent (Invitrogen) at a ratio of 1:3.75
(w:v) in 4 ml DMEM for 30 min, and then added to the cells. Six h later, the media was
replaced with fresh DMEM/7.5% FBS. After 48 h, cells were pelleted by centrifugation
at 650 x g for 5 min at 4 oC, washed with 10 ml homogenization buffer consisting of 250
mM sucrose/50 mM Tris pH 7.4/0.25 mM CaCl2, 10 μl benzamidine (200 mg/ml) and
protease inhibitors, and then cells were collected by centrifugation at 800 x g for 5 min at
4 oC. Collected cell pellets were layered with 10 ml homogenization buffer consisting of
250 mM sucrose/50 mM Tris pH 7.4/0.25 mM CaCl2, 10 μl benzamidine (200 mg/ml),
and protease inhibitors, and kept at -80 oC until required for membrane vesicle
preparation (Section 2.7).
2.7 Preparation of Membrane Vesicles from MRP1 Transfectants
Membrane vesicles were prepared from transfected HEK293T cells as previously
described (Loe et al., 1996a; Letourneau et al., 2005). Briefly, cell pellets were thawed in
warm water for 5 min, and while on ice, resuspended in homogenization buffer
(described in Section 2.6). Cells were exploded by argon cavitation (300 psi, 4 oC, 5 min),
39
and the exploded cell suspension was centrifuged at 800 x g at 4 oC for 10 min in an IEC
CentraGP8R centrifuge (DJB Labcare Ltd., Buckinghamshire, UK). The supernatant (15
ml) was layered onto an equal volume of a 35% (w/v) sucrose/1 mM EDTA/50 mM Tris,
pH 7.4 cushion. After centrifugation at 100,000 x g at 4 oC for 60 min in an OptimaTM L90K Ultracentrifuge (Beckman Coulter, Fullerton, CA), the interface was removed and
placed in a 25 mM sucrose/50 mM Tris, pH 7.4 buffer solution and centrifuged at
100,000 x g at 4 oC for 30 min again. The membranes were washed with 1 ml TrisSucrose Buffer (TSB) (250 mM sucrose, 50 mM Tris, pH 7.4), and centrifuged at 68,000
x g at 4 oC for 20 min in an TL-100 Ultracentrifuge (Beckman Coulter, Fullerton, CA).
The pellet was resuspended in 50-100 μl TSB by passage through a 1 ml syringe 12 times
with a 27-gauge needle, aliquoted (14 μg protein per aliquot), and stored at -80oC until
required. Vesicular protein concentrations were determined using the Bradford protein
assay (Bio-Rad) and a standard curve prepared with BSA.
2.8 Determination of MRP1 Protein Levels in Transfected Cells
The expression levels of wild-type and MRP1 mutants (and untransfected HEK293T
controls) were determined by immunoblot analysis of membrane vesicle proteins from
transfected cells as described previously (Ito et al., 2001a). Proteins were loaded onto a
1.5 mm thick SDS-polyacrylamide gel (7% acrylamide resolving, 4% acrylamide
stacking) with a 15-well comb using Laemmli buffer containing 14 nM 2mercaptoethanol, separated by electrophoresis at 75-150 V for 1.5-2 h, and then
electrotransferred (100 V, 1 h) to a PVDF membrane using a BioRad Mini-Gel apparatus.
The PVDF membrane was washed (3 times, 5 min per wash) at room temperature with
40
TBS-T, and blocked with 4% (w/v) skim milk in TBS-T for 30 min at room temperature,
followed by incubation with the human MRP1-specific murine MAb QCRL-1 (1:10000
in 4% skim milk/TBS-T, 4 °C, overnight) (Hipfner et al., 1996). Immunoblots were
washed (3 times, 5 min per wash) with TBS-T and incubated with horseradish
peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:10000 in 4% skim
milk/TBS-T, room temperature, 1 h). Blots were washed (5 times, 5 min per wash) with
TBS-T, followed by application of equal volumes (1.5 ml each) of Western Lightning
Plus-Enhanced Chemiluminescence (ECL) blotting substrates, and then the membrane
was exposed to film within 15 min of ECL addition, for 1-5 s exposures. Relative levels
of MRP1 expression were measured by densitometric analysis of the blots using ImageJ
1.36b software (http://rsb.info.nih.gov/ij/). Binding of rabbit anti-Na+/K+ ATPase (diluted
1:5000 in 4% skim milk/TBS-T, and incubated at 4 °C overnight) followed by
horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (diluted
1:10000 in 4% skim milk/TBS-T at room temperature for 1 h) was used to control for
protein loading. Washes and ECL substrate were applied as described for MRP1 protein
detection. After densitometry of the Na+/K+ ATPase bands, relative MRP1 expression
levels were determined to take into account differences in total protein loaded.
2.9 MRP1-mediated Transport of 3H-Labeled Substrates by Membrane Vesicles
ATP-dependent vesicular transport of 3H-labeled substrates by membrane vesicles was
measured using a microplate rapid filtration technique as previously described
(Letourneau et al., 2005), with the following modification: 20 nCi of [3H]LTC4 was used
per reaction instead of 10 nCi. For LTC4 transport assays, reactions were performed in
41
96-well round bottom plates (Sarstedt, Newton, NC) at 23 °C (water bath) in a 30 µl
reaction volume containing 50 nM [3H]LTC4 (20 nCi), 4 mM AMP or ATP, 10 mM
MgCl2, 10 mM creatine phosphate, 100 µg/ml creatine kinase, and 2 µg of vesicle protein.
Creatine phosphate and creatine kinase were omitted from reactions containing AMP, and
volumes were made up with TSB. Reaction mixtures (24 µl) and membrane vesicles (2
µg in 6 µl) were allowed to acclimate (3 min) to the reaction temperature separately prior
to transport. Uptake was stopped after 1 min by addition to a Dynablock 2000TM 96-deepwell plate (VWR International, Mississauga, ON) containing ice-cold TSB (800 µl), and
the diluted incubation mixture was filtered through a UniFilter GF/B 96-well filter plate
(Packard, Meriden, CT). Radioactivity was quantified using liquid scintillation counting
by a TopCountNXTTM Microplate Scintillation and Luminescence Counter (PerkinElmer). Transport in the presence of AMP was subtracted from transport in the presence
of ATP to determine ATP-dependent [3H]LTC4 uptake. Uptake of E217βG was similarly
measured, except that membrane vesicles (2 µg of protein) were incubated at 37 °C
(water bath) in a total reaction volume of 30 µl containing [3H]E217βG (400 nM; 20 nCi)
and components as described for [3H]LTC4 transport.
2.10 Kinetic Analysis of [3H]E217βG Transport
Kinetic analyses were performed as previously described (Letourneau et al., 2005), with
some modifications. Km and Vmax values for [3H]E217βG transport were determined by
measuring uptake at eight different E217βG concentrations (0.25-25 µM) and either 2 or
4 µg protein for 1 min at 37 °C in a 30 µl reaction volume of transport buffer containing
components as described above. For the three highest E217βG concentrations (5, 10, 25
42
µM), 25 nCi [3H]E217βG was used. Data were analyzed, and kinetic parameters were
calculated by nonlinear regression using GraphPad PrismTM 3.0 (GraphPad Software, La
Jolla, CA).
2.11 Photolabeling of MRP1 by [3H]LTC4
Photolabeling of MRP1 was performed as previously described (Koike et al., 2004,
Letourneau et al., 2008). Briefly, membrane vesicles (50 µg protein in 40 µl TSB) were
incubated with [3H]LTC4 (200 nM; 100 nCi) in a 96-well flat bottom flexible plate (BD
Biosciences, Durham, NC) at room temperature for 30 min and then snap-frozen by
placing into liquid nitrogen for 10 sec. Membrane vesicles (1.5 µg protein) were retained
prior to cross-linking, and immunoblotted to quantify relative MRP1 expression.
[3H]LTC4 was cross-linked to MRP1 by irradiating (1100 µW, 302 nm) for ten 1 min
exposures using a CL-1000 Ultraviolet Crosslinker (DiaMed, Mississauga, ON), with
snap-freezing in liquid nitrogen for 10 sec between exposures. Radiolabeled proteins (40
µg) were loaded on a 1.5 mm thick SDS-polyacrylamide gel (7% acrylamide resolving,
4% acrylamide stacking) with a 10-well comb using Laemmli buffer containing 14 nM 2mercaptoethanol, and separated by electrophoresis at 75-150 V (1.5-2 h). The gel was
processed for autoradiography by first incubating in a fixing solution (65:25:10; water,
isopropanol, acetic acid) for 30 min, and then incubation in AmplifyTM solution for 30
min. The gel was dried on a Model 583 Gel Dryer (Bio-Rad) in a vacuum at 80 °C for 24 h. After drying, the gel was exposed to Kodak X-Omat LS autoradiography film at 80 °C for 5 days or longer, as required. Cassettes containing film were allowed to
43
acclimate to room temperature (3 h) prior to processing. Signal intensities on the film
were analyzed by densitometry using ImageJ 1.36b software as described above.
2.12 Statistical Analyses
Differences in [3H]E217βG and [3H]LTC4 transport activities of wild-type and mutant
MRP1 were analyzed for statistical significance using a 2-tailed Student’s t-test
(GraphPad Prism 3.0). Differences were considered significant when p < 0.05.
44
CHAPTER III: FUNCTIONAL ANALYSIS OF
GLU1253, GLU1255, GLU1262 AND GLU1266 MRP1 MUTANTS
3.1 Introduction
Ionizable amino acids (Glu, Asp, Arg, and Lys) are present within internal regions of
hydrophobic proteins as a result of evolutionary processes (e.g. variation and selection),
and it appears that these amino acids randomly accumulated (through mutations) within
the protein’s enzymatic or active site, in order to develop the ability to perform a specific
task (Isom et al., 2008). This suggests that ionizable amino acids are likely to have an
important role in modulating the function of such proteins. Consistent with this notion,
previous studies on MRP1 have demonstrated that many ionizable amino acids are
sensitive to mutation when present in or proximal to TMs of the core structure (MSD1
and MSD2) of the transporter (Deeley and Cole, 2006; Conseil et al., 2007, 2009; Maeno
et al., 2009). For example, it has been shown that conservative and non-conservative
substitutions of Lys332 and His335 in TM6 cause substrate selective changes in activity,
such that LTC4 transport is eliminated or reduced while E217βG transport remains intact
(Haimeur et al., 2002, 2004). On the other hand, even conservative mutations of Asp336 in
TM6, and Arg1249 at the TM17-cytoplasmic interface completely eliminate activity
(Haimeur et al., 2004; Situ et al., 2004). Non-conservative mutation of another ionizable
amino acid, Asp430 in the cytoplasmic loop connecting TM7 to TM8, sharply reduces
expression of MRP1 (Haimeur et al., 2004).
In the present study, acidic amino acids were identified in the TM17-proximal
region of MRP1, and their role in expression and transport investigated (Figure 3.1).
45
A
MSD0
TM
1
2
3
4
M SD1
5
6
7
8
M SD2
9 10 11
1 2 13 1 4 15 1 6 17
H 2N
COOH
NBD2
1266
1262
1255
1253
1248
1228
NBD1
1227 AGLVGLSVSYSLQVTTYLNWL VRMSS EMETNIVAV ERLKEYSE 1269
B
A la 1 227
V a l 1 248
A rg 1 249
G lu 1 269
Figure 3.1: Secondary structure, and in silico illustration of the TM17-proximal
region of MRP1.
A, a secondary structure of MRP1 showing the predicted locations of TM17 (black
cylinder) and its COOH-proximal region (black star), defined in this study as amino acids
1228-1248 and 1249-1269, respectively. Key conserved residues are shown in bold. B,
the predicted locations of TM17 (dark gray) and its COOH-proximal region (black)
spanning amino acids 1228 to 1269 are shown in the three-dimensional homology model
of MRP1 (lacking MSD0) generated using the crystal structure of Sav1866 from S.
aureus as template (DeGorter et al., 2008). The lipid bilayer is represented by dotted
lines.
46
Because of its proximity to the functionally important TM17, and because it links TM17
to NBD2, it was hypothesized that ionizable residues in this region could be important for
MRP1 function. In addition, since current Sav1866-based models of MRP1 predict that
this α-helical region extending from TM17 is likely to participate in interhelical
interactions, it was further hypothesized that amino acids in the TM17-proximal helix
might form electrostatic interactions with residues extending from TM14 and TM15. In
this chapter, the results of experiments designed to test these hypotheses are described.
3.2 Results
3.2.1 Secondary Structure Predictions, Sequence Alignments, and In Silico
Illustrations g of the region COOH-proximal to TM17
To begin this study (and those described in Chapter IV), several algorithms (DSC, MLRC,
PHD, Predator) were used to determine a consensus secondary structure of the region
COOH-proximal to TM17 (defined in this study as amino acids 1249-1269) (Figure 3.1)
as described in Chapter II (Section 2.2). The consensus structure obtained identifies
residues 1249-1269 as being α-helical in nature, which is in agreement with the Sav1866based homology model of the core structure of MRP1 (DeGorter et al., 2008) (Figure
3.2A). Next, a multiple sequence alignment of TM17 and the COOH-proximal region of
MRP1 (residues 1228-1269) with the sequences of its eleven mammalian homologs was
performed using ClustalW v1.0 (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html). As illustrated in Figure 3.2B,
TM17 and its COOH-proximal region are well conserved, and four acidic residues
(Glu1253, Glu1255, Glu1262, and Glu1266) within the COOH-proximal region exhibit a high
47
1249
DSC
MLRC
PHD
Predator
R
h
h
e
h
M
h
h
e
h
S
h
h
e
c
S
c
h
e
c
E
c
h
h
c
M
h
h
h
c
E
h
h
h
h
T
h
h
h
h
N
h
h
h
h
I
h
h
h
h
V
h
h
e
h
A
h
h
h
h
V
h
h
h
h
E
h
h
h
h
R
h
h
h
h
12
66
12
62
12
53
12
55
A
L
h
h
h
h
K
h
h
h
h
E
h
h
h
h
Y
h
h
h
h
S
h
h
c
h
E
c
h
c
h
T
c
c
c
h
E
c
c
c
h
K
c
c
c
h
E
c
c
c
c
A
c
c
c
c
PWQ
c c c
c c e
c c e
c c c
I
c
e
e
e
Q
c
c
c
e
E 1280
c
c
c
e
Sec. Cons. h h h c ? h h h h h h h h h h h h h h h ? c c c c c c c ? e c c
B
MRP1/ABCC1
MRP3/ABCC3
MRP2/ABCC2
MRP6/ABCC6
SUR1/ABCC8
SUR2/ABCC9
MRP7/ABCC10
MRP8/ABCC11
MRP9/ABCC12
MRP5/ABCC5
MRP4/ABCC4
CFTR/ABCC7
Sav1866
1227 A
P
G
A
A
S
P
Y
T
P
A
E
G
G
D
G
G
G
G
S
S
A
G
G
L
L
T
L
L
L
L
F
S
Y
Q
R
V
V
V
V
V
V
V
K
K
A
V
V
G
G
G
G
G
G
G
V
G
G
G
G
L
L
F
F
L
L
L
M
L
L
L
I
S
S
V
S
G
G
S
A
D
A
A
I
V
V
L
V
L
L
L
V
L
I
L
L
S
S
S
S
T
L
S
N
S
S
S
T
Y
Y
N
A
Y
Y
Y
I
Y
Y
Y
L
S
S
A
A
A
A
A
V
I
A
A
A
L
L
L
L
L
L
L
L
I
V
L
M
Q
Q
N
Q
M
T
S
Q
Q
Q
T
N
V
V
I
V
V
I
L
L
L
L
L
I
T
T
T
T
S
T
T
A
S
T
M
M
T
F
Q
Q
N
N
G
S
G
G
G
S
Y
A
T
T
Y
Y
L
S
L
L
M
T
L
L
L
L
L
L
L
F
L
F
F
L
N
N
N
Q
N
N
S
Q
Q
Q
Q
Q
W
W
W
W
W
W
G
A
V
F
W
W
L
M
L
V
M
V
L
T
C
T
C
A
V
I
V
V
V
V
V
A
V
V
V
V
R
R
R
R
R
R
S
R
R
R
R
N
M
M
M
N
N
N
S
I
T
L
Q
S
S
M
T
W
L
L
F
G
G
A
S
S
S
S
S
T
A
A
T
L
T
S
A
I
E
D
E
D
D
D
Q
E
E
E
E
D
M
L
I
L
M
L
T
T
T
T
V
V
E
E
E
E
E
E
E
E
Q
E
E
D
T
S
T
N
L
V
A
A
A
A
N
S
N
N
N
S
Q
Q
M
Q
K
R
M
L
I
I
I
I
L
M
L
F
F
F
M
M
V
V
V
V
G
G
V
T
T
T
I
R
A
A
A
S
A
A
S
A
S
S
S
S
V
V
V
V
V
V
V
V
V
V
V
V
ERL
ERV
ERI
ERM
KRI
KKV
ERL
ERI
ELL
ERI
ERV
SRV
1266
1262
1255
1253
1248
1228
α-helix
K
K
T
Q
H
N
E
L
R
N
I
F
E
E
E
D
G
S
E
Q
E
H
E
K
Y
Y
Y
Y
L
F
Y
Y
Y
Y
Y
F
S
S
T
A
L
L
T
M
I
I
T
I
E 1269
K
K
W
K
T
C
K
S
K
D
D
VGTLAAFVGYLELLFGPLR R LVASFTTLTQSFASMDRVFQLID
Figure 3.2: Structural predictions, and sequence alignments of the TM17-proximal
region of MRP1.
A, secondary structure predictions of amino acids 1249-1280 of human MRP1. h, helical;
c, coiled; e, extension. B, sequence alignment of human MRP1 (residues 1227-1269) and
its eleven homologs, as well as the sequence of Sav1866, generated using ClustalW 1.0.
Amino acids which are identical in a majority of homologs are shown on a black
background, while those which are conserved are shown on a gray background. G1228
and V1248 define the boundaries of TM17, while residues E1253, E1255, E1262, and
E1266 are of key interest in this study. SUR, sulfonylurea receptor; CFTR, cystic fibrosis
transmembrane conductance regulator.
48
degree of sequence identity and/or similarity (Glu1253, >90% similarity; Glu1255, >80%
identity, >90% similarity; Glu1262, 75% identity, 75% similarity; and Glu1266, >55%
similarity).
As described in Chapter I (Section 1.6), atomic homology models of the fourdomain core structure of nucleotide-bound MRP1 (MRP1.B99990092.pdb) were recently
generated in our lab using the crystal structure of Sav1866 from S. aureus as template
(DeGorter et al., 2008). Using a model based on a multiple sequence alignment of MRP1
homologs, the locations and side-chain positions of the four conserved Glu residues
relative to a putative substrate translocation pathway formed by MSD1 and MSD2 were
examined. According to this model, the side-chain of Glu1253 projects into the putative
translocation pathway, while the side-chains of Glu1255, Glu1262, and Glu1266 project away
from it (Figure 3.3).
When the potential for interhelical electrostatic, H-bond, and hydrophobic
interactions between TM17-proximal amino acids and possible partner residues in
adjacent α-helices was examined, four amino acids complementary (i.e. oppositely
charged or polar, and capable of forming electrostatic and hydrogen bonds) to the sidechains of the four Glu residues were found, with each pair estimated to be within 3.4-9.7
Å of each other’s side-chains (discussed below in Section 3.3) (Table 3.1). Thus, the four
pairs of potentially interacting amino acids identified were: Glu1253 and Lys1141, Glu1255
and Arg1138, Glu1262 and Arg1142 and Glu1266 and Arg1075 (Figure 3.4), of which two were
investigated experimentally as described below (Section 3.2.6).
49
E1253
E1255
E1262
E1266
Figure 3.3: Putative relative locations and side-chain positions of Glu1253, Glu1255,
Glu1262 and Glu1266 in a homology model of MRP1.
The three-dimensional homology model of MRP1 (lacking MSD0) of DeGorter et al.
(2008) described in Chapter I (Section 1.6) showing the predicted location of TM17proximal region (dark gray), as well as the predicted locations and side-chain positions of
Glu1253, Glu1255, Glu1262 and Glu1266 (in black) (amino acids are shown as ‘spheres’ in the
full model, and as ‘sticks’ in the inset). E1253 projects into the translocation pathway,
while E1255, E1262, and E1266 project away from it.
50
TM17-Proximal Residue
Potential Partner Residue
(Position in MRP1)
Predicted Distance
Between Closest SideChain Atoms (Å)
E1253
K1141 (TM15-proximal)
9.7
E1255
R1138 (TM15-proximal)
6.1
E1262
R1142 (TM15-proximal)
3.4
E1266
R1075 (TM14-proximal)
3.4
Table 3.1: Estimated distances between side-chains of Glu1253, Glu1255, Glu1262 and
Glu1266, and other amino acids which potentially form interhelical bonds in MRP1.
Predicted distances between the four conserved Glu residues in the TM17-proximal helix
and complementary amino acids in the TM14- and TM15-proximal helices were
estimated from a Sav1866-based atomic homology model of MRP1 (DeGorter et al.,
2008), as described in the text.
51
A
B
C
D
Figure 3.4: In silico illustrations of potential paired interhelical interactions
involving side-chains of Glu1253 and Lys1141, Glu1255 and Arg1138, Glu1262 and Arg1142,
and Glu1266 and Arg1075.
Using a homology model of MRP1 (lacking MSD0) (DeGorter et al., 2008), the predicted
locations of TM17-proximal residues (black) and their potential interhelical partners
(dark gray) are shown. Also shown are potential electrostatic interactions between Glu1253
and Lys1141 (A), Glu1255 and Arg1138 (B), Glu1262 and Arg1142 (C), and Glu1266 and Arg1075
(D). Dotted lines represent distances between side-chains, and these values are listed in
Table 3.1.
52
3.2.2 Expression, and E217βG and LTC4 Transport Activities of Ala-Substituted
Glu1253, Glu1255, Glu1262, and Glu1266 MRP1 Mutant Proteins
To begin analysis of the functional importance of these four Glu residues, single Alasubstituted mutant constructs were created by site-directed mutagenesis and transfected
into HEK293T cells. Immunoblot analyses, first of whole cell lysates to confirm
transfection efficiency (not shown), and subsequently, of membrane vesicles prepared
from the transfected HEK293T cells were performed. Wild-type and mutant MRP1
proteins were detected using the human MRP1-specific MAb (QCRL-1) (Hipfner et al.,
1994; Ito et al., 2001a). As shown in Figure 3.5A, all four mutants were expressed at
levels comparable to wild-type MRP1 in membrane vesicles, demonstrating that Alasubstitution of these residues does not affect the biosynthesis of MRP1.
Next, the ability of the four Ala-substituted Glu MRP1 mutants to mediate ATPdependent transport of E217βG and LTC4 was examined using a microplate in vitro
vesicular uptake assay (Loe et al., 1996; Letourneau et al., 2005). As shown in Figure
3.5B&C, the E1253A MRP1 mutant showed a statistically significant loss of both
E217βG and LTC4 transport activity (p < 0.05). However, the loss of E217βG transport
(by 75%) was substantially more than the loss of LTC4 transport (by 30%), suggesting
some substrate selective effects of this mutation. The E1262A mutant also showed a
statistically significant loss of both E217βG and LTC4 transport activities (p < 0.05);
however, the loss of LTC4 transport (by 75%) was greater than the loss of E217βG
transport (by 50%). In contrast to E1253A and E1262A LTC4 transport activities, LTC4
transport by E1255A was similar to that of wild-type MRP1, with only a small but
significant (p < 0.05) increase in E217βG uptake (by 20%), while LTC4 transport by
53
Figure 3.5: Expression levels and vesicular uptake of 3H-labeled organic anions by
Ala-substituted Glu1253, Glu1255, Glu1262 and Glu1266 MRP1 mutants.
A, Shown is a representative immunoblot of membrane vesicles (0.5 and 1.0 μg protein)
prepared from HEK293T cells transfected with E1253A, E1255A, E1262A, E1266A, and
wild-type (WT) MRP1 cDNA expression vectors. Untransfected cells were used as a
negative control (control). MRP1 was detected with MAb QCRL-1, and the relative
(corrected based on Na+/K+ ATPase loading controls) protein expression levels are shown
below the blot and were determined by densitometry as described in Chapter II (Section
2.8). B and C, ATP-dependent uptake of [3H]E217βG (B) and [3H]LTC4 (C) was
measured in the membrane vesicles shown in panel A. Vesicles prepared from
untransfected cells were used as a negative control (control). Uptake values were
normalized based on mutant MRP1 levels relative to WT-MRP1 levels (according to
panel A), and uptake by the mutants was expressed as a percent of uptake by WT-MRP1.
The results shown are the means (+S.D.) of three or more independent experiments; n
values are shown above each bar. Similar results were obtained in at least one additional
experiment using vesicles prepared from an independent transfection. MV, membrane
vesicles; *, significantly different from wild-type MRP1 activity (p < 0.05).
54
A
C
M V (μg)
0 .5
on
1 .0
tr o
l
W
0 .5
T -M
1 .0
RP
1
E
0 .5
5
12
1 .0
3A
E
0 .5
5
12
1 .0
5A
E
0 .5
6
12
1 .0
2A
E
0 .5
6
12
6A
1 .0
MRP1
N a + /K + A T P a s e
-
R e la tive e xp re s s io n
(c o rre c te d )
B
-
1 .0
1 .0
1 .4
1 .1
1 .2
(% WT-MRP1)
E217βG Uptake
150
1 .1
1 .0
1 .1
1 .0
0 .9
*
(3)
(9)
(3)
100
*
(5)
*
50
(5)
*
(9)
C
E1
26
6A
E1
26
2A
E1
25
5A
E1
25
3A
C
on
tro
l
W
TM
R
P1
0
(3)
(10)
100
*
(3)
*
(5)
50
*
(5)
*
(10)
0
C
on
tro
l
W
TM
R
P1
E1
25
3A
E1
25
5A
E1
26
2A
E1
26
6A
(% WT-MRP1)
LTC4 Uptake
150
55
the E1266A mutant was slightly (20%) but significantly decreased (p < 0.05) and E217βG
uptake was comparable to wild-type MRP1. Since the changes in the transport activities
of the E1255A and E1266A mutants were so small as to be unlikely biochemically
significant, these mutants were not investigated further.
3.2.3 Expression and Transport Activities of MRP1 Mutants with Same- and OppositeCharge Substitutions of Glu1253 and Glu1262
To determine if it is the negative charge at positions 1253 and 1262 that is important for
supporting MRP1 transport, and if a larger positively charged side-chain would cause a
greater disruption of transport activity, same and opposite charge substitutions of Glu1253
(E1253D and E1253K) and Glu1262 (E1262D and E1262R) were generated and expressed
in HEK293T cells. Immunoblotting experiments revealed that MRP1 protein expression
levels of these mutants in both whole cell lysates (not shown) and membrane vesicles
(Figures 3.6A&B) were similar to wild-type MRP1. The same-charge E1253D MRP1
mutant displayed E217βG and LTC4 transport activities comparable to those of wild-type
MRP1, while the opposite-charge E1253K mutant exhibited substantially and
significantly reduced E217βG and LTC4 uptake (decreased by 85% and 75%,
respectively; p < 0.05) (Figures 3.6C&D). In contrast, the same-charge E1262D MRP1
mutant exhibited significantly reduced E217βG and LTC4 uptake (decreased by 50% and
80%, respectively) (p < 0.05) (Figures 3.6C&D). Transport by the opposite-charge
E1262R mutant was also significantly reduced to a similar degree, by 75% for E217βG,
and by 90% for LTC4 uptake (p < 0.05) (Figures 3.6C&D).
56
Figure 3.6: Expression levels and vesicular uptake of 3H-labeled organic anions by
same- and opposite-charge substitutions of Glu1253 and Glu1262 MRP1 mutants.
A and B, Shown are representative immunoblots of membrane vesicles (0.5 and 1.0 μg
protein) prepared from HEK293T cells transfected with wild-type (WT) MRP1, as well
as E1253A/D/K (A) and E1262A/D/R (B) mutant MRP1 expression vectors. MRP1 was
detected with MAb QCRL-1, and an antibody against Na+/K+ ATPase was used as a
loading control, as described in the legend of Figure 3.5. C and D, ATP-dependent uptake
of [3H]E217βG (C) and [3H]LTC4 (D) was measured in membrane vesicles prepared from
HEK293T cells transfected with WT-MRP1, and Glu1253 and Glu1262 mutant MRP1
(E1253A/D/K, E1262A/D/R) cDNAs, as described in the legend to Figure 3.5. The
results shown are the means (+S.D.) of three or more independent experiments; n values
are shown above each bar. Similar results were obtained in at least one additional
experiment using vesicles prepared from an independent transfection. MV, membrane
vesicles; *, significantly different from wild-type MRP1 activity (p < 0.05).
57
A
C
MV (μg)
0.5
on
l
tr o
1.0
W
0.5
T-
M
RP
1.0
1
E
0.5
5
12
1.0
3A
E
0.5
5
12
1.0
3D
0.5
2
E1
53
K
1.0
MRP1
Na + /K + ATPase
-
Relative expression
(corrected)
-
B
Co
MV (μg)
0.5
1.0 1.0 1.3 1.0 1.0 1.0 1.5 1.1
o
n tr
1.0
l
W
0.5
T-
M
1.0
RP
1
0.5
2
E1
62
1.0
A
2
E1
0.5
62
1.0
D
0.5
2
E1
62
R
1.0
MRP1
Na + /K + ATPase
Relative expression
(corrected)
C
-
-
1.0 1.0 0.7 0.8 0.6 0.9 0.7 0.9
E217βG Uptake
(% WT-MRP1)
150
(11)
(3)
100
*
(3)
*
(5)
50
*
(5)
*
(5)
*
(4)
*
(11)
C
on
tr o
W
l
TM
R
P1
E1
25
3A
E1
25
3D
E1
25
3K
E1
26
2A
E1
26
2D
E1
26
2R
0
D
(11)
(3)
100
*
(5)
50
*
*
*
(3)
(5)
(3)
*
*
(5)
(11)
2R
26
E1
26
2D
2A
E1
26
3K
58
E1
25
E1
25
3D
3A
E1
25
P1
E1
W
TM
R
tro
l
0
C
on
LTC4 Uptake
(% WT-MRP1)
150
A ‘revertant’ construct "E(D)1262E", in which the E1262D construct was
mutated back to the wild-type sequence, was generated and characterized to rule out the
possibility that the phenotype of E1262D mutant may be due to mutations introduced
inadvertently outside of the cloning region during mutagenesis. However, this was not the
case as expression levels of E(D)1262E in membrane vesicles were comparable to wildtype MRP1 (not shown), and LTC4 transport activity of this mutant was also comparable
to wild-type MRP1 (not shown).
3.2.4 Kinetic Analysis of [3H]E217βG Uptake by E1253A, E1262A and E1262D MRP1
Mutants
To determine whether reduced E217βG uptake by the Ala-substituted Glu1253 and Glu1262,
and Asp-substituted Glu1262 MRP1 mutants was caused by differences in apparent uptake
affinity (Km) or changes in the maximum transport velocity (Vmax), kinetic parameters
were determined by measuring uptake for a fixed time period (1 min) over a range of
E217βG concentrations (0.25-25 µM). Experiments using 4 µg protein per point were
conducted as previously described (Conseil et al., 2006), while experiments using 2 µg
protein per point were included since it was determined that this amount of protein would
be sufficient for these analyses (Letourneau et al., 2005). Kinetic parameters were then
determined by non-linear regression analysis of the data, and the variability of Km and
Vmax values between experiments may be due to the range in the date of preparation (or
‘freshness’) of membrane vesicles used to analyze E217βG kinetics.
While there were differences in the absolute Km values for E217βG uptake by the
E1253A and E1262A mutants, in that each was substantially increased (3.1–14.3-fold and
59
A
(pmol/mg protein/min)
E217βG Uptake
400
300
200
100
0
B
„ W T-MRP 1
z E 1253A
0
5
(pmol/mg protein/min)
15
20
25
E 2 1 7 β G (μ M)
400
E217βG Uptake
10
300
200
100
0
„ W T-MRP1
z E1262A
0
5
10
15
20
25
E 2 17β G (μ M)
C
E217βG Uptake
(pmol/mg protein/min)
300
200
100
0
„
z
0
5
10
15
W T- M RP 1
E 1262D
20
25
E 2 1 7 β G (μ M )
Figure 3.7: Kinetic analysis of [3H]E217βG uptake by Ala-substituted Glu1253 and
Glu1262, and Asp-substituted Glu1262 MRP1 mutants.
ATP-dependent [3H]E217βG uptake by membrane vesicles prepared from HEK293T cells
transfected with wild-type MRP1, Ala-substituted Glu1253 (A), and Ala- (B) and Aspsubstituted Glu1262 (C) MRP1 mutants were measured at the indicated E217βG
concentrations (0.25-25 μM) for 1 min at 37 oC, as previously described (Conseil et al.,
2006). Values shown are duplicate determinations in a single representative experiment
(2 μg protein/point); 2-3 independent experiments were performed for each mutant (see
Table 3.2). Vmax values are corrected for any differences in expression of the mutant
proteins relative to wild-type MRP1. Kinetic parameters for E217βG transport were
determined from non-linear regression analysis and are summarized in Table 3.2.
60
E217βG Transport
Vmax (pmol/mg/min)
Vmax/Km x 103
(mg/l/min)
Km (µM)
(μg protein/pt)
2
4
2
4
2
4
WT-MRP1
351
194
499
2.1
6.7
4.0
0.17
0.03
0.12
E1253A
454
202
1368
10.7
20.7
57.0
0.04
0.01
0.02
WT-MRP1
306
499
2.4
4.0
0.13
0.12
E1262A
240
270
7.2
7.2
0.03
0.04
WT-MRP1
300
350
N/D
5.4
12.5
N/D
0.06
0.03
N/D
E1262D
134
104
N/D
6.2
10.3
N/D
0.02
0.01
N/D
Table 3.2: Summary of kinetic parameters of [3H]E217βG uptake by Ala-substituted
Glu1253 and Glu1262, and Asp-substituted Glu1262 MRP1 mutants.
Kinetic analyses were performed by measuring E217βG uptake (initial concentration
range of 0.25-25 µM) for 1 min at 37 oC using either 2 or 4 μg vesicle protein per point as
explained in text. Each Km and Vmax value shown is from a single independently prepared
batch of vesicles and is the mean of duplicate determinations. Vmax values have been
corrected for differences in protein expression relative to WT-MRP1. Vmax/Km x 103 ratios
were calculated and indicate the relative E217βG uptake efficiency of the MRP1 mutants.
N/D, not determined.
61
1.8–3.0-fold, respectively) relative to wild-type MRP1, the Vmax values of both mutants
were comparable to wild-type MRP1 (Figure 3.7 & Table 3.2). On the other hand, while
the Km (E217βG) of E1262D was similar to wild-type MRP1, the Vmax value was reduced
(by 55-70%) (Figure 3.7 & Table 3.2). Thus, this indicates that charge is not important
for E217βG uptake affinity in this case, but instead for uptake efficiency (as reflected by
the 3–6-fold difference in the Vmax/Km x 103 ratio). As reported in Table 3.2, the observed
uptake efficiencies of E1253A, E1262A, and E1262D were reduced by 65-85%, 65-75%,
and 65%, respectively, compared to wild-type MRP1.
The kinetic data correlate, reasonably well with the E217βG transport data.
E217βG transport by the E1253A mutant was decreased by 75%, while the apparent Km
(E217βG) was 3.1–14.3-fold higher than wild-type MRP1. Transport by the E1262A
mutant was reduced by 50%, and the apparent Km (E217βG) was 1.8–3.0-fold higher than
wild-type MRP1. Finally, E217βG transport by the E1262D mutant was reduced by 50%
compared with wild-type MRP1, and this was associated with a 55-70% reduction in Vmax.
Thus, reductions in Km (E217βG) of E1253A and E1262A explain their decreased
E217βG transport, while the reduced Vmax of E1262D explains its decreased E217βG
transport.
3.2.5 Photolabeling of E1253A, E1262A and E1262D MRP1 Mutants by [3H]LTC4
To determine if the reduced LTC4 uptake by E1253A, E1262A, and E1262D MRP1
mutants was associated with changes in substrate binding, [3H]LTC4 photolabeling
experiments were conducted. As shown in Figure 3.8, E1253A and E1262A were
photolabeled by [3H]LTC4 at levels comparable to wild-type MRP1, this is somewhat
62
unexpected since the apparent Km (E217βG) was reduced and it was anticipated that
binding of LTC4 would also be reduced in concordance with the reduced LTC4 transport
by these mutants. On the other hand, photolabeling of the E1262D MRP1 mutant was
approximately 40% lower (Figure 3.8), which was unexpected since the Km (E217βG)
was comparable to wild-type MRP1, and thus it was anticipated that [3H]LTC4 labeling
by this mutant would be similar to wild-type MRP1.
3.2.6 Expression and E217βG and LTC4 Transport Activities of K1141E/E1253K and
R1142E/E1262R Double Reciprocal MRP1 Mutants
One way to explore the possibility that two residues might form an interhelical bond is to
determine whether double exchange (reciprocal) mutants of the paired amino acids would
restore wild-type activity. To do this, single TM15-proximal K1141E and R1142E, as
well as the double mutants K1141E/E1253K and R1142E/E1262R were needed. The
K1141E and R1142E mutant constructs were available from a previous study (Conseil et
al., 2006), while the double exchange mutants were generated as described in Chapter II
(Section 2.4).
Immunoblot analyses demonstrated that the expression levels of the K1141E and
R1142E MRP1 mutants in whole cell lystates (not shown) and membrane vesicles
(Figures 3.9A & 3.10A, respectively) were similar to wild-type MRP1. Expression levels
of K1141E observed in this study were substantially higher (by 3.3–4.8-fold) than
previously reported (Conseil et al., 2006), a difference that cannot presently be explained.
63
P1
l
D
A
A
R
o
r
62
53
t
62
M
2
n
2
2
T
E1
Co
E1
E1
W
A
Relative [3 H]LTC4
photolabeling
-
1.0
0.8
0.8
0.7
Corrected [3 H]LTC4
photolabeling
-
1.0
0.9
1.1
0.6
l
tro
n
Co
B
MV (μg)
0.5
1.0
0.5
W
P1
R
M
T-
1.0
0.5
[3 H]LTC4
labeled
MRP1
A
A
D
62
53
62
2
2
2
1
1
E
E
E1
1.0
0.5
1.0
0.5
1.0
MRP1
Na+/K+ ATPase
Relative expression
-
-
1.0
1.0
1.2
1.4
0.4
0.6
0.8
1.2
Corrected expression
-
-
1.0
1.0
0.7
1.0
0.6
0.9
1.0
1.3
Figure 3.8: Photolabeling of Ala-substituted Glu1253 and Glu1262, and Aspsubstituted Glu1262 MRP1 mutant proteins by [3H]LTC4.
A, Membrane vesicles (50 µg protein) were incubated with [3H]LTC4 and irradiated
(1100 µW, 302 nm). After resolving vesicles (40 µg protein) by SDS-PAGE, the gel was
processed for autoradiography and exposed to film (7 day exposure). Signal intensities on
the film were analyzed by densitometry. Relative levels of [3H]LTC4 photolabeling are
indicated below the image, and have been corrected for any differences in protein
expression levels relative to wild-type MRP1, as shown by the immunoblot in panel B.
The [3H]LTC4 photolabeling results shown are from a single experiment. Similar results
were observed in a second independent experiment. B, Shown is an immunoblot which
quantifies the relative protein expression levels of the MRP1 mutants used for the
photolabeling in Panel A. MRP1 was detected with MAb QCRL-1, and an antibody
against Na+/K+ ATPase was used as a loading control, as described in the legend to
Figure 3.5. The mean MRP1 expression levels from 0.5 and 1.0 μg protein/lane were
used to correct for differences in photolabeling. HEK293T cells were used as a control in
all photolabeling and immunoblot experiments.
64
Figure 3.9: Expression levels and vesicular uptake of 3H-labeled organic anions by
MRP1 mutants containing single and exchange substitutions of Lys1141 and Glu1253.
A, Shown is a representative immunoblot of membrane vesicles (0.5 and 1.0 μg protein)
prepared from HEK293T cells transfected with K1141E, E1253K, K1141E/E1253K, and
wild-type (WT) MRP1 cDNA expression vectors. MRP1 was detected with MAb QCRL1, and an antibody against Na+/K+ ATPase was used as a loading control, as described in
the legend to Figure 3.5. B and C, ATP-dependent uptake of [3H]E217βG (B) and
[3H]LTC4 (C) was measured in membrane vesicles prepared from HEK293T cells
transfected with WT-MRP1, and single and exchange substitutions of Lys1141/Glu1253.
The results shown are the means of two independent experiments. Untransfected cells
were used as a control in all experiments. MV, membrane vesicles.
65
A
Co
MV (μg)
0.5
E
P1
E/
K
E
R
1
3
1
M
14
25
14
T1
1
1
K
E
K
W
ol
n tr
1.0
0.5
1.0
0.5
1.0
0.5
1.0
0.5
53
12
K
1.0
MRP1
Na+/K+ ATPase
Relative expression
(corrected)
B
-
-
1.0 1.0 1.3 1.9 2.0 1.3 1.6 1.3
(% WT-MRP1)
E217βG Uptake
150
(2 )
100
(2 )
50
(2 )
(2 )
(2 )
W
C
on
t
ro
l
TM
R
P1
K1
14
1E
E1
K1
25
14
3K
1E
/E
12
53
K
0
C
(2)
100
50
(2)
(2)
(2)
(2)
P1
K1
14
1E
E1
K1
25
14
3K
1E
/E
12
53
K
W
TM
R
ro
l
0
C
on
t
(% WT-MRP1)
LTC4 Uptake
150
66
Figure 3.10: Expression levels and vesicular uptake of E217βG and LTC4 by MRP1
mutants containing single and double exchange substitutions of Arg1142 and Glu1262.
A, Shown is a representative immunoblot of membrane vesicles (0.5 and 1.0 μg protein)
prepared from HEK293T cells transfected with R1142E, E1262R, R1142E/E1262R, and
wild-type (WT) MRP1 cDNA expression vectors. MRP1 was detected with MAb QCRL1, and an antibody against Na+/K+ ATPase was used as a loading control, as described in
the legend to Figure 3.5. B and C, ATP-dependent uptake of [3H]E217βG (B) and
[3H]LTC4 (C) was measured in membrane vesicles prepared from HEK293T cells
transfected with WT-MRP1, and single and exchange substitutions of Arg1142/Glu1262.
The results shown are the means (+S.D.) of 3-4 independent experiments. Similar results
were obtained in at least one additional experiment using vesicles prepared from an
independent transfection. Untransfected cells were used as a control in all experiments.
MV, membrane vesicles; *, significantly different from wild-type MRP1 activity (p <
0.05).
67
A
Co
MV (μg)
0.5
P1
E
R
R
M
42
62
1
14
2
T1
1
W
R1
R
E
ol
ntr
1.0
0.5
1.0
0.5
1.0
0.5
1.0
0.5
26
1
/E
2E
2R
1.0
MRP1
Na+/K+ ATPase
-
Relative expression
(corrected)
B
-
1.0 1.0 0.9 0.8 0.9 1.0 0.7 0.9
(% WT-MRP1)
E217βG Uptake
150
(3)
100
*
( 3* )
(3)
*
50
(3)
*
(3)
W
C
on
t
ro
l
TM
R
P1
R
11
42
E
E1
R
11
26
42
2R
E/
E1
26
2R
0
C
(4)
100
( 4* )
50
*
(4)
*
(4)
*
(4)
on
tro
W
l
TM
R
P1
R
11
42
E
E
R
12
11
62
42
R
E/
E1
26
2R
0
C
(% WT-MRP1)
LTC4 Uptake
150
68
When assessed using a vesicular uptake assay, E217βG uptake by both the K1141E (Fig.
3.9B) and R1142E (Fig. 3.10B) mutants was reduced (by 80% and 40%, respectively) (p
< 0.05), as was uptake of LTC4 (by 80% each) (p < 0.05) (Figures 3.9C & 3.10C,
respectively). These uptake levels were comparable to those previously reported (Conseil
et al., 2006), and confirm that Lys1141 and Arg1142 are important for MRP1 function.
Double exchange (reciprocal) mutations (K1141E/E1253K and R1142E/E1262R)
were then generated as described in Chapter II (Section 2.4). Expression levels of the
K1141E/E1253K mutant in whole cell lysates (not shown) and membrane vesicles
(Figure 3.9A) were comparable to wild-type MRP1. However, when the ability of
K1141E/E1253K reciprocal MRP1 mutant to mediate ATP-dependent vesicular uptake of
E217βG and LTC4 was examined, only a modest increase (by 20-30%) in E217βG uptake
was observed compared to the single K1141E and E1253K mutants, and they still
remained substantially lower than that of wild-type MRP1 (Figure 3.9B). LTC4 uptake by
the double reciprocal mutant was also comparable to that of the two single mutants
(Figures 3.9C).
Expression levels of the R1142E/E1262R reciprocal mutant were also similar to
that of wild-type MRP1 in whole cell lysates (not shown) as well as membrane vesicles
(Figure 3.10A). However, E217βG uptake by R1142E/E1262R was increased by only 3545% relative to uptake by E1262R, and unchanged relative to R1142E (Figure 3.10B),
and remained significantly (p < 0.05) decreased (by 65-75%) relative to wild-type MRP1.
LTC4 uptake by this reciprocal MRP1 mutant was improved (by 15-25%) (p < 0.05)
compared to R1142E and E1262R (Figure 3.10C), although it remained significantly
reduced (by 60%) (p < 0.05) relative to wild-type MRP1 (Figure 3.10C).
69
3.3 Discussion
In this study, Ala substitutions were initially made to determine which of the four Glu
residues in the TM17-proximal cytoplasmic region of MRP1 were important for
expression and substrate transport. Replacing Glu with Ala is a so-called ‘cavity-creating’
mutation due to the small volume of the Ala side-chain. The side-chain of Ala is also
incapable of H-bonding due to its lack of hydrogen donor or acceptor electrons. Thus,
Ala provides a non-conservative substitution for polar, aromatic, and ionizable amino
acids, as well as a conservative substitution (with decreased steric volume) for non-polar
amino acids. For these reasons, Ala-substitution is commonly used in structure-function
studies utilizing site-directed mutagenesis.
From the biochemical analysis described in Section 3.2.2, it is clear that Alasubstitution of the four Glu residues does not affect MRP1 levels, suggesting that these
acidic amino acids are not critical for proper folding and assembly during its biosynthesis
in HEK293T cells (Figure 3.5A). However, Ala-substitution of Glu1253 and Glu1262
significantly reduced transport of both E217βG and LTC4 (Figure 3.5B&C). E217βG
kinetic analyses suggest that decreased apparent uptake affinities (increases in apparent
Km) by both E1253A and E1262A contribute to the reduction in E217βG transport activity
(Table 3.2), while in seeming contrast [3H]LTC4 photolabeling experiments showed that
the decreases in LTC4 transport by the E1253A and E1262A mutants were not due to
detectable differences in MRP1 labeling by this substrate (Figure 3.8A). The similarity of
the E1253A and E1262A phenotypes could suggest that the two Glu residues play a
similar role in substrate translocation. However, if this was true, then one would expect
that same charge substitutions of the two residues would have the same effect, but this
70
was not the case. Thus, substitution of Glu1253 with Asp had no affect on expression or
transport activity, indicating the role of Glu1253 can be retained by Asp, despite its shorter
side-chain length (Figure 3.6C&D). In contrast, however, transport activity was not
retained with an Asp-substitution at position 1262 (Figure 3.6C&D). Thus, the roles of
these two amino acids must differ. Finally, substitution of an oppositely (positively)
charged amino acid at both positions (1253 and 1262) significantly decreased transport
activities, a result that might have been expected because such non-conservative
substitutions of Glu1253 and Glu1262 are much more likely to disrupt MRP1 structure and
thus its function (Figure 3.6C&D).
Although the apparent Km (E217βG) of the E1262D mutant was similar to wildtype MRP1, the Vmax was reduced by approximately 55-70% (Table 3.2). However,
[3H]LTC4 labeling of E1262D was also reduced (by 40%) (Figure 3.8A). This suggests
that although binding of E217βG by E1262D is intact, the transport efficiency was
impaired, whereas reductions in LTC4 uptake (by 80%) by E1262D appear to be
explained, at least in part, by a decreased apparent association with this substrate. Thus,
the volume of the Glu1262 side-chain seems important for MRP1 function, but in different
ways for E217βG and LTC4 transport. Similar findings have been reported for the human
concentrative nucleoside transporter 3 (hCNT3), in which Asp-substitution of Glu519 was
able to restore functionality of Na+ but not H+ coupling compared with non-conservative
mutations, suggesting subtly different positional requirements for this negative charge
(Slugoski et al., 2009). Therefore, it may be concluded that MRP1-Glu1262 plays a
complex role in substrate binding/efflux, and could contribute to a highly structured local
environment.
71
It is of interest to note that several of the residues chosen for functional analysis in
this study are of clinical relevance in homologous ABCC proteins, including CFTR
(ABCC7) and MRP6 (ABCC6). Mutation of residues in CFTR analogous to Glu1253 and
Glu1255 of MRP1 (Asp1152 and Asp1154, respectively) have been linked to cystic fibrosis,
and like the Glu residues in MRP1, are located in the cytoplasmic extension connecting
the last TM to NBD2 (Mussaffi et al., 2006; Vankeerberghen et al., 1998). The CFTR
mutation of D1152H has been shown to cause a mild disease state, possibly by
interference with chloride channel gating (Mussaffi et al., 2006). In addition, although the
CFTR mutation D1154G has been found in a patient with cystic fibrosis, interpretation of
the phenotype is somewhat confounded by the fact that the patient also has the wellcharacterized and disease-causing ΔF508 mutation on the other allele (Vankeerberghen et
al., 1998).
These observations together with the present MRP1 data indicate that mutation of
an amino acid in the region connecting the most COOH-proximal TM to NBD2 in at least
one other ABCC protein, namely TM12 in CFTR, which is analogous to one of the
TM17-proximal residues of MRP1 sensitive to mutation (i.e. Glu1253), is associated with
disease. Based on these findings, the functional importance of this conserved amino acid
may suggest that the structure of this region is conserved as well.
In contrast to CFTR, only one mutation in MRP6/ABCC6 (analogous to Glu1266 in
MRP1) is associated with Pseudoxanthoma elasticum, and was found by searching the
literature, including the PXE international foundation website (http://www.pxe.org), and
the ABCC6 human gene mutation database (http://www.hgmd.cf.ac.uk/ac/all.php).
Mutation of Asp1238 to His was found in one individual with Pseudoxanthoma elasticum,
72
but this mutation has not yet been well characterized (Ladewig et al., 2006). In addition,
it must be noted that it is currently unclear how mutations in MRP6 cause PXE, which is
in contrast to mutations in MRP1 and CFTR, where the physiological substrates of these
ABCC proteins are well known.
Charged residues within hydrophobic regions of soluble proteins have been
shown to interact with oppositely charged residues in order to neutralize charge and
minimize energetic constraints (Anderson et al., 1990; Tissot et al., 1996). This charge
stabilization also holds true for membrane proteins, and this phenomenon can
substantially contribute to the global functionality of such proteins (Zhou et al., 1994).
Pairs of residues that may potentially interact via ionic bonding (i.e. salt bridge
formation) and H-bonding were identified (Table 3.1), and since substitution of Glu1253
and Glu1262 with Ala had the greatest effects on transport activity, the potential
interhelical pairs involving these two residues were investigated by reciprocal
mutagenesis.
The reported maximum distance between two residues involved in a salt bridge
(ionic bond) is ~3.5 Å (Chang et al., 2008; Sackin et al., 2009). However, a previous
study in our lab (Haimeur and Cole, unpublished) obtained biochemical evidence
suggesting that an interhelical bond occurs via an electrostatic interaction between Lys396
and Asp436, despite the fact that these two residues are estimated to be 10.5 Å apart in
current MRP1 models. Thus, reciprocal mutagenesis experiments showed that the double
mutant K396D/D436K was a fully functional transporter, even though the single mutants
K396D and D436K exhibited substantially reduced (by >70%) E217βG and LTC4
transport activities. These observations suggest a relatively exclusive or dominant
73
interaction between these two amino acids. Therefore, in the present study it seemed
reasonable to set a cutoff value, with respect to the maximal distance between two
residues, that is higher than that which may be theoretically achievable. In this way,
imprecision in the model with regard to placement of the side-chains can be allowed for.
Thus, reciprocal mutagenesis experiments were undertaken despite the fact that Glu1253 is
predicted to be within 9.7Å of its potential bonding partner Lys1141 (Table 3.1).
The E1253K MRP1 mutant exhibited significant reductions (>75%) in both
E217βG and LTC4 transport (Figure 3.9B&C), and so by mutating the nearby Lys1141 to
Glu (K1141E), it was hoped that transport activity might be restored as a result of
acid/base pairing. However, this was not the case since only a slight increase in E217βG
uptake (to 40% of wild-type MRP1 activity) was observed (Figure 3.9B&C). These
observations do not exclude the possibility that Glu1253 interacts with Lys1141, but do
indicate that the interaction is not exclusive, or is somewhat weak.
With respect to Glu1262, it is interesting to note that the amino acid corresponding
to this residue in the distantly related ABCC protein SUR2 (ABCC9) is a Lys (1280)
residue, and the amino acid in SUR2 at the same position as Arg1142 in MRP1 (its
potential bonding partner) is a Glu (1162) residue. The presence of oppositely charged
residues at the same two positions of these proteins in which an interhelical interaction
has been predicted to occur in MRP1 is intriguing and could suggest that an
evolutionarily conserved interhelical salt bridge might exist in both proteins (i.e. Arg1142
and Glu1262 in MRP1, and Glu1162 and Lys1280 in SUR2). However, the positioning of
these charged residues could just be a coincidence, since one might expect that if a
conserved salt bridge existed, it would be present in more than just one homolog.
74
The E1262R MRP1 mutant exhibited substantial reductions in both E217βG and
LTC4 transport (Figure 3.10B&C), and so by mutating the nearby Arg1142 to Glu
(R1142E), and restoring the possibility of acid/base pairing by creating R1142E/E1262R,
it was thought that transport function could be enhanced. However, this was not the case,
suggesting Glu1262 likely does not interact exclusively with Arg1142 (Figure 3.10B&C).
Together, the data presented here indicate that the side-chain positions of Glu1253
and Lys1141, and Glu1262 and Arg1142 may be accurately predicted in the homology model
of MRP1, and thus are indeed too far apart to provide a substantial interhelical bond,
and/or that interhelical bonding interactions are more complex than hypothesized.
75
CHAPTER IV: FUNCTIONAL ANALYSIS OF ALA-SUBSTITUTED
VAL1261, ARG1263 AND TYR1267 MRP1 MUTANTS
4.1 Introduction
As described and discussed in Chapters I and III, many functionally important amino
acids in the TM helices of MRP1 are ionizable and/or polar (Haimeur et al., 2002; Situ et
al., 2004; Ren et al., 2002). Aromatic residues in the TM helices of MRP1 also play a
role in MRP1 function, such that the transport, and in some cases, the binding of
substrates is altered when these residues are mutated (Ito et al., 2001a; Koike et al., 2002;
Zhang et al., 2002; Campbell et al., 2004). For example, Trp1246 in TM17 is critical for
E217βG transport, in that both conservative and non-conservative substitutions of this
residue abrogate E217βG uptake, while LTC4 transport remains intact (Ito et al., 2001a).
In contrast, the functional importance of aliphatic amino acids located within or proximal
to TM helices has not yet been explored. In the present study, the aim was to investigate
the importance of three highly conserved aliphatic, basic, and aromatic residues (Val1261,
Arg1263, and Tyr1267, respectively) in the α-helical region COOH-proximal to TM17, in
determining the expression and transport activity of MRP1 (Figure 3.1). Because of its
proximity to the functionally important TM17, and because it links TM17 to NBD2, it
was hypothesized that conserved amino acids in this region could be important for MRP1
function. In addition, since current Sav1866-based models of MRP1 predict that this αhelical region extending from TM17 is likely to participate in interhelical interactions, it
was further hypothesized that these amino acids in the TM17-proximal helix might form
electrostatic interactions with residues extending from TM14, as well as those within the
76
cytoplasmic loop (CL6) connecting TM13 to TM14. In this chapter, the results of
experiments designed to test the importance of individual amino acids are described, and
residues possibly involved in interhelical bonds are identified.
4.2 Results
4.2.1 Secondary Structure Predictions, Sequence Alignments, and In Silico
Illustrations of the Region COOH-Proximal to TM17
As described in Chapter III (Section 3.2) and shown in Figure 4.1, secondary structure
predictions and multiple sequence alignments of the cytoplasmic TM17-proximal region
of MRP1 and its eleven homologs revealed the presence of a significant number of highly
conserved amino acids. In addition to the 4 Glu residues investigated in Chapter III, three
other amino acids (Val1261, Arg1263, and Tyr1267) within the COOH-proximal region of
TM17 of MRP1 exhibited a high degree of sequence identity and/or similarity (Val1261,
100% identity; Arg1263, >80% identity, >90 similarity; and Tyr1267, 75% identity, >90%
identity), and these were targeted for investigation.
According to a model of the four-domain core structure of MRP1 (DeGorter et al.,
2008), the side-chains of Val1261 and Arg1263 project toward the cytoplasmic α-helical
extension of TM14, while the side-chain of Tyr1267 projects toward CL6 (Figure 4.2).
Thus, none of these three amino acids are predicted to project directly into the putative
translocation pathway. When Val1261, Arg1263, and Tyr1267, and amino acids in adjacent αhelices were examined in the MRP1 homology model, the following pairs of potential
bonding interactions were identified: Val1261 with Val1083 (van der Waals), Arg1263 with
77
1249
DSC
MLRC
PHD
Predator
12
67
12
6
12 1
63
A
R
h
h
e
h
M
h
h
e
h
S
h
h
e
c
S
c
h
e
c
E
c
h
h
c
M
h
h
h
c
E
h
h
h
h
T
h
h
h
h
N
h
h
h
h
I
h
h
h
h
V
h
h
e
h
A
h
h
h
h
V
h
h
h
h
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h
h
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h
R
h
h
h
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L
h
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h
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h
h
Y
h
h
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h
S
h
h
c
h
E
c
h
c
h
T
c
c
c
h
E
c
c
c
h
K
c
c
c
h
E
c
c
c
c
A
c
c
c
c
P
c
c
c
c
WQ
c c
c e
c e
c c
I
c
e
e
e
Q
c
c
c
e
E 1280
c
c
c
e
Sec. Cons. h h h c ? h h h h h h h h h h h h h h h ? c c c c c c c ? e c c
B
Sav1866
G
G
D
G
G
G
G
S
S
A
G
G
L
L
T
L
L
L
L
F
S
Y
Q
R
V
V
V
V
V
V
V
K
K
A
V
V
G
G
G
G
G
G
G
V
G
G
G
G
L
L
F
F
L
L
L
M
L
L
L
I
S
S
V
S
G
G
S
A
D
A
A
I
V
V
L
V
L
L
L
V
L
I
L
L
S
S
S
S
T
L
S
N
S
S
S
T
Y
Y
N
A
Y
Y
Y
I
Y
Y
Y
L
S
S
A
A
A
A
A
V
I
A
A
A
L
L
L
L
L
L
L
L
I
V
L
M
Q
Q
N
Q
M
T
S
Q
Q
Q
T
N
V
V
I
V
V
I
L
L
L
L
L
I
T
T
T
T
S
T
T
A
S
T
M
M
T
F
Q
Q
N
N
G
S
G
G
G
S
Y
A
T
T
Y
Y
L
S
L
L
M
T
L
L
L
L
L
L
L
F
L
F
F
L
N
N
N
Q
N
N
S
Q
Q
Q
Q
Q
W
W
W
W
W
W
G
A
V
F
W
W
L
M
L
V
M
V
L
T
C
T
C
A
V
I
V
V
V
V
V
A
V
V
V
V
R
R
R
R
R
R
S
R
R
R
R
N
M
M
M
N
N
N
S
I
T
L
Q
S
S
M
T
W
L
L
F
G
G
A
S
S
S
S
S
T
A
A
T
L
T
S
A
I
E
D
E
D
D
D
Q
E
E
E
E
D
M
L
I
L
M
L
T
T
T
T
V
V
E
E
E
E
E
E
E
E
Q
E
E
D
T
S
T
N
L
V
A
A
A
A
N
S
N
N
N
S
Q
Q
M
Q
K
R
M
L
I
I
I
I
L
M
L
F
F
F
M
M
V
V
V
V
G
G
V
T
T
T
I
R
A
A
A
S
A
A
S
A
S
S
S
S
V
V
V
V
V
V
V
V
V
V
V
V
ERL
ERV
ERI
ERM
KRI
KKV
ERL
ERI
ELL
ERI
ERV
SRV
126 7
126 3
1227 A
P
G
A
A
S
P
Y
T
P
A
E
126 1
MRP1/ABCC1
MRP3/ABCC3
MRP2/ABCC2
MRP6/ABCC6
SUR1/ABCC8
SUR2/ABCC9
MRP7/ABCC10
MRP8/ABCC11
MRP9/ABCC12
MRP5/ABCC5
MRP4/ABCC4
CFTR/ABCC7
124 8
122 8
α-helix
K
K
T
Q
H
N
E
L
R
N
I
F
E
E
E
D
G
S
E
Q
E
H
E
K
Y
Y
Y
Y
L
F
Y
Y
Y
Y
Y
F
S
S
T
A
L
L
T
M
I
I
T
I
E 1269
K
K
W
K
T
C
K
S
K
D
D
VGTLAAFVGYLELLFGPLR R LVASFTTLTQSFASMDRVFQLID
Figure 4.1: Structural predictions and sequence alignments of Val1261, Arg1263, and
Tyr1267 of MRP1.
A, secondary structure predictions of amino acids 1249-1280 of human MRP1. H,
helical; C, coiled; E, extension. B, sequence alignment of human MRP1 (residues 12271269) and its eleven homologs, as well as the sequence of Sav1866, generated using
ClustalW 1.0. Amino acids which are identical in a majority of homologs are shown on a
black background, while those which are conserved are shown on a gray background.
G1228 and V1248 define the boundaries of TM17, while residues V1261, R1263, and
Y1267 were examined in this study. SUR, sulfonylurea receptor; CFTR, cystic fibrosis
transmembrane conductance regulator.
78
V1261
R1263
Y1267
Figure 4.2: Putative relative locations and side-chain positions of Val1261, Arg1263,
and Tyr1267 in a homology model of MRP1.
A three-dimensional homology model of the four-domain core structure of MRP1 shows
the predicted location of TM17-proximal region (dark gray) spanning amino acids 1249
to 1269, as well as the predicted locations and side-chain positions of Val1261, Arg1263,
and Tyr1267 (in black) (amino acids are shown as ‘spheres’ in the full model, and as
‘sticks’ in the inset). The side-chains of Val1261 and Arg1263 project toward the TM14proximal α-helix, while the side-chain of Tyr1267 projects toward CL6.
79
Glu1079 (ionic), and Tyr1267 with Phe1063 (π -π/aromatic stacking) (Figure 4.3 and Table
4.1). It should be noted that the predicted distance (5.3 Å) between the side-chains of
Tyr1267 and Phe1063 is larger than that likely to make the above bonding interaction
plausible (Chang et al., 2008; Sackin et al., 2009). However, this potential interaction
was still investigated, taking into account the notion that there may be imprecision in
MRP1 models with respect to placement of the side-chains of these amino acids.
4.2.2 Expression and E217βG and LTC4 Transport Activities of Ala-Substituted Val1261,
Arg1263, and Tyr1267 MRP1 Mutant Proteins
To begin analysis of the functional importance of Val1261, Arg1263, and Tyr1267, single
Ala-substitutions of these residues were made, and after transfection in HEK293T cells
immunoblot analysis of whole cell lysates (not shown) and membrane vesicles was
performed using the human MRP1-specific MAb (QCRL-1) as before (Hipfner et al.,
1994; Ito et al., 2001a). As shown in Figure 4.4A, all three mutants were expressed at
levels comparable to wild-type MRP1 in membrane vesicles, demonstrating that Alasubstitution of these residues did not affect the biosynthesis of MRP1 in HEK293T cells.
Next, the ability of these three MRP1 mutants to mediate ATP-dependent
transport of E217βG and LTC4 was examined using a microplate in vitro vesicular uptake
assay (Loe et al., 1996; Letourneau et al., 2005). As before, uptake levels were
normalized to take into account differences in expression of the mutant proteins relative
to wild-type MRP1, and activity was calculated as a percent of the activity of vesicles
enriched for wild-type MRP1.
80
A
B
C
Figure 4.3: In silico illustrations of potential interhelical interactions involving sidechains of Val1261 and Val1083, Arg1263 and Glu1079, and Tyr1267 and Phe1063.
A homology model of the core structure (MSD1 and MSD2) of MRP1 shows the
predicted location of the cytoplasmic TM17-proximal amino acids (black) and their
potential interhelical bonding partners (dark gray). Shown are potential interactions
between the side-chains of Val1261 and Val1083 (A), Arg1263 and Glu1079 (B), and Tyr1267
and Phe1063 (C). Dotted lines represent the distances between the side-chains, which are
listed in Table 4.1.
81
TM17-Proximal Residue
Potential Partner Residue
(Position in MRP1)
Predicted Distance
Between Closest SideChain Atoms (Å)
V1261
V1083 (TM14-proximal)
2.9
R1263
E1079 (TM14-proximal)
3.2
Y1267
F1063 (CL6)
5.3
Table 4.1: Estimated distances between side-chains of Val1261, Arg1263 and Tyr1267,
and other amino acids in other helices which might form bonding interactions.
The distances between the side-chains of the indicated pairs of amino acids were
estimated based on a Sav1866-based atomic homology model of MRP1 (DeGorter et al.,
2008) using PyMOL (DeLano Scientific; http://www.pymol.org).
82
Figure 4.4: Expression levels and vesicular uptake of E217βG and LTC4 by Alasubstituted Val1261, Arg1263, and Tyr1267 MRP1 mutants.
A, Shown is a representative immunoblot of membrane vesicles (0.5 and 1.0 μg protein)
prepared from HEK293T cells transfected with V1261A, R1263A, Y1267A, and wildtype (WT) MRP1 cDNA expression vectors. Untransfected cells were used as a negative
control (control). MRP1 was detected with MAb QCRL-1, and relative MRP1 expression
levels were adjusted to take into account differences in total protein loaded using an
antibody against Na+/K+ ATPase. Relative MRP1 levels (corrected for total protein
loaded) are shown below the blot, and were determined by densitometry as described in
Chapter II (Section 2.8). B and C, ATP-dependent uptake of [3H]E217βG (B) and
[3H]LTC4 (C) was measured in membrane vesicles prepared from HEK293T cells
transfected with WT-MRP1, and Ala-substituted mutant MRP1 cDNA expression vectors.
Vesicles prepared from untransfected cells were used as a negative control (control).
Uptake values were normalized based on mutant MRP1 levels relative to WT-MRP1
levels (according to panel A), and uptake by the mutants was expressed as a percentage
of uptake by WT-MRP1. The results shown are the means (+S.D.) of at least three
independent experiments (numbers in parentheses above the bars). Similar results were in
at least one additional experiment using vesicles prepared from an independent
transfection (not shown). MV, membrane vesicles; *, significantly different from wildtype MRP1 activity (p < 0.05).
83
P1
A
A
l
R
A
o
63
61
tr
67
-M
2
2
n
2
T
V1
R1
W
Y1
Co
A
MV (μg)
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
MRP1
Na+/K+ ATPase
Relative expression
(corrected)
B
-
-
1.0 1.0 1.1 1.1 1.1 0.9 1.0 1.0
(% WT-MRP1)
E217βG Uptake
150
(4)
*
100
(4)
*
*
(4)
(4)
50
*
(4)
Y1
26
7A
12
63
A
R
V1
26
1A
R
M
TW
C
on
tro
l
P1
0
C
*
(3)
(3)
100
*
(3)
50
*
(3)
*
(3)
V1
26
1A
R
12
63
A
Y1
26
7A
R
P1
W
TM
on
tro
l
0
C
(% WT-MRP1)
LTC4 Uptake
150
84
As shown in Figures 4.4B&C, and in contrast to the Ala-substituted Glu1253 and
Glu1262 mutants described in Chapter III (Figure 3.5), the Ala-substituted Val1261, Arg1263
and Tyr1267 mutants showed only modest reductions (by 25-55%) in transport activity,
although these were statistically significant (p < 0.05). However, the loss of E217βG
activity (by 30%) by the R1263A mutant was somewhat less than the loss of LTC4
transport (by 55%), suggesting some substrate selective effect of this mutation, since
these activities were statistically different from each other (p < 0.05). For the V1261A
and 1267A mutants, the reduction in transport of LTC4 and E217βG was ≤ 35%.
4.3 Discussion
A Sav1866-based MRP1 homology model was initially used to predict the putative
locations of the highly conserved Val1261, Arg1263, and Tyr1267 in the TM17-proximal
region of MRP1. According to this model, the side-chains of Val1261 and Arg1263 project
toward the TM14-proximal α-helix, while the side-chain of Tyr1267 projects toward CL6.
Their putative orientations projecting away from the predicted translocation pore could
suggest that any changes in activity observed after Ala-substitution of these residues
would likely not be due to direct interaction of substrate with these amino acids.
When tested experimentally, Ala-substitution of Val1261, Arg1263, and Tyr1267 did
not affect MRP1 biosynthesis (Figure 4.4A), indicating that these amino acids are not
critical for proper folding and stable expression of MRP1 in HEK293T cells. Vesicular
uptake assays showed that Ala-substitution of Val1261, Arg1263, and Tyr1267 reduced both
the E217βG and LTC4 transport activities by a modest (by 25-55%), but statistically
significant degree (Figure 4.4B&C). These findings suggest that the side-chain
85
orientations of these amino acids may in fact be accurately predicted, since mutations did
not substantially affect transport of either E217βG or LTC4, thus indicating that these
residues likely do not directly interact with substrates.
In the homologous CFTR, several mutations of the amino acid analogous to
Arg1263 of MRP1 have been reported (http://www.hgmd.cf.ac.uk/ac/all.php). Thus, the
CFTR mutation R1162L (located in the cytoplasmic extension connecting TM12 to
NBD2) was found in one individual with cystic fibrosis, although this patient also had
another diseasing-causing mutation, G542X (Groman et al., 2002). In addition, a R1162Q
mutation was found in another individual with a mild disease state (i.e. having normal
sweat chloride levels and pancreatic sufficiency) (Strandvik et al., 2001). Unfortunately,
these findings do not indicate whether or not mutation of CFTR-Arg1162 alone is
detrimental to the function of this protein.
Mutations of residues in CFTR analogous to MRP1-Tyr1267 have not been
reported. On the other hand, mutations of the analogous MRP6-Tyr1239 were found in the
ABCC6/MRP6 human gene mutation database (http://www.hgmd.cf.ac.uk/ac/all.php).
The Y1239H mutation in TM17 of MRP6 was detected in an individual diagnosed late (at
32 years of age) with mild PXE (Schulz et al., 2005).
In summary, mutation of amino acids analogous to MRP1-Arg1263 and Tyr1267 in
the region connecting the most COOH-proximal TM to NBD2 in the ABCC proteins
CFTR and MRP6 are associated with disease, although mild in some cases. Furthermore,
it is clear the prevalence of these mutations in patients with cystic fibrosis and PXE is
low.
86
CHAPTER V: CONCLUSIONS AND FUTURE DIRECTIONS
Homology models of MRP1 based on the crystal structure of Sav1866 have been useful
in guiding the design and aiding in the interpretation of biochemical analyses of this
transporter. In this thesis, MRP1 homology models were used to aid in the interpretation
of biochemical data for single mutants of seven conserved residues in the region COOHproximal to TM17 of MRP1, and assisted in the design of experiments involving double
exchange mutants of several of these TM17-proximal amino acids. Implications of these
data with regard to MRP1 function and structure, as well as conclusions and future
directions are discussed in this chapter.
5.1 Implications for MRP1 Structure
As discussed in Chapter III, it has been shown that Ala-substitution of Glu1255 and Glu1266
had little effect on the transport activity of MRP1, which is consistent with a model in
which both residues are predicted to project away from the translocation pathway of
MRP1. On the other hand, Glu1253 was found to be important for the translocation of both
E217βG and LTC4, although to varying degrees (i.e. more so for E217βG). These
observations together with its predicted projection into the translocation pathway are
compatible with, but do not prove the idea that this amino acid directly interacts with at
least E217βG. These findings are in agreement with several previous observations in
which mutation of Arg1249 and Met1250 in the TM17-proximal region also greatly reduced
transport of both organic anions (Situ et al., 2004; Haimeur et al., unpublished), while
mutation of Thr1242, Asn1245, and Trp1246 within TM17 selectively affect the transport of
87
glucuronide conjugates (Zhang et al., 2001; Zhang et al., 2002; Ito et al., 2001a; Leslie et
al., 2001a).
In contrast, although Glu1262 is predicted to project away from the putative
translocation pathway, Ala- and Asp-substitutions significantly reduced transport of both
E217βG and LTC4, and Ala-substitution of Glu1262 reduced the apparent uptake affinity
(Km) of E217βG. These data may indicate that the homology model does not sufficiently
predict the true side-chain conformation of Glu1262, since this amino acid has
demonstrated its importance for apparent uptake affinity with E217βG, suggesting the
side-chain may in fact project into the pore. Therefore, the side-chain orientation of
Glu1262 in the atomic homology model of MRP1 used in this study may need to be refined
as a result of these findings. A direct interaction with LTC4 has not yet been thoroughly
investigated for the Ala- and Asp-substituted Glu1262 mutants, and thus kinetic analyses
of LTC4 uptake are needed to support the notion that the true side-chain orientation
projects into the translocation pathway. Furthermore, the side-chain orientation of this
amino acid may differ during the various conformations of MRP1 during its catalytic
cycle, and thus indirect effects may contribute to the observed phenotypes (e.g. apparent
reductions in E217βG affinity), and therefore the model may in fact be accurate to some
extent.
In addition, these findings may suggest that Glu1262 is important for transducing
signals between MSD2 and NBD2, since mutations caused an overall reduction in
transport activity. One possibility is that mutation of Glu1262 affects the geometry of
NBD2 to which TM17 is connected, and in this way, affects the ATPase activity of the
transporter. This has been suggested for certain amino acids in TM12 of P-gp (Crowley et
88
al., 2009). Thus, the mutations V988C and Q990C in TM12 of P-gp appears to reduce
ATP hydrolysis by perturbing the geometry of the helical extension which is connected to
NBD2, as demonstrated by in silico characterization using the crystal structure of murine
P-gp (Aller et al., 2009). Similarly, TM17-proximal mutations that change NBD2 of
MRP1 might alter dimerization of its NBDs, as well as the subsequent interaction of ATP
at the NBSs. In this way, the coupling of ATP binding and hydrolysis with transport
would be affected, and thus an overall reduction in activity might be expected. Further
experiments using azio-derivatives of
32
P-ATP to determine the ability of E1253A,
E1262A, and E1262D mutants to bind and hydrolyze ATP are needed to test this idea
(Letourneau et al., 2008).
The reciprocal mutagenesis studies described in Chapter III indicate that Glu1253
and Lys1141, and Glu1262 and Arg1142 do not interact exclusively with each other, since
double exchange mutations were unable to enhance MRP1 transport activity. It is quite
possible that the mutations disrupted other important bonding interactions, and therefore
these data should be considered inconclusive with respect to the hypothesized interhelical
interactions. Therefore, the side-chain orientations of these residues may in fact be
accurately predicted in the homology model of MRP1, and thus the cutoff distance of 9.7
Å set in this study likely overestimated the maximum interhelical distance in which an
interaction can occur, and so a shorter cutoff (e.g. 3-5 Å) would have been sufficient.
Based on the conserved nature of Val1261, Arg1263 and Tyr1267, it was initially
thought that each residue may play a role in MRP1 structure and function. However, data
described in Chapter IV demonstrate that these amino acids are not critical for plasma
membrane expression, and had little influence on MRP1 function compared to Glu1253
89
and Glu1262. On the other hand, Ala-substitution of Arg1263 did have some effect on
transport, which is in agreement with all other studies to date that indicate a functional
role for most ionizable residues in MRP1. Overall, these observations are thus far
consistent with current MRP1 models, which predict the side-chain orientations of Val1261,
Arg1263 and Tyr1267 away from the translocation pore.
5.2 Limitations of MRP1 Homology Models and Reciprocal Mutagenesis
Although much of the data described in this thesis are compatible with the predicted
projections of the seven amino acids studied by current models of MRP1 (DeGorter et al.,
2008), the use of homology models is still limited by the fact that the template used to
generate them is a homodimeric bacterial ABC transporter structure (Dawson and Locher,
2006). Differences in membrane composition of eukaryotic and prokaryotic cells have
been established, and thus may pose some challenges with respect to modeling eukaryotic
transporters from prokaryotic structures (Costerton et al., 1974).
A limitation of reciprocal mutagenesis is that this technique relies on amino acids
in a potential interacting pair to interact essentially exclusively with each other (Zhou et
al., 1994; Chang et al., 2008). Although this type of interaction is possible, it is more
likely that the side-chains of residues which assist in maintaining the structure of
polytopic membrane proteins, including MRP1, interact with multiple atoms in nearby
amino acids (Pace, 2009). An alternative and relatively common approach to studying the
proximity between TM helices, and thus potentially interacting amino acid pairs, is to use
a Cys-less mutant, as has been done for P-gp and other membrane proteins (Loo and
Clarke, 1995; Taylor et al., 2001). By using a mutant protein that lacks Cys residues, Cys
90
residues can be introduced at positions which are thought to be involved in interhelical
bonding interactions, and following treatment with a sulfide crosslinking agent, pairs of
amino acids that form exclusive bonds in the native protein can be identified by
electrophoretic methods (Loo et al., 2004). However, this approach may be problematic
for MRP1, since Cys substitutions of several of the 25 Cys residues it contains have
significantly affected the structural and functional properties of this transporter (Yang et
al., 2002; Leslie et al., 2003b).
5.3 Concluding Remarks
In summary, the data presented in this thesis suggest the functional importance of several
conserved amino acids (mainly Glu1253 and Glu1262) in the TM17-proximal region of
MRP1, although none of the seven residues investigated are critical for expression. Thus,
Glu1253 and Glu1262 have different and complex roles in substrate recognition and
translocation, while no functional importance can be ascribed to Glu1255 and Glu1266. The
data suggest that Val1261, Arg1263 and Tyr1267 may be important for MRP1 function as
well.
Glu1262 could be further investigated by analyses of E1262D, to determine
whether its reduced photolabeling of [3H]LTC4 is due to changes in apparent LTC4
uptake affinitiy (Km). Investigations involving the mutation-sensitive Glu1253 and Glu1262
could include determining if the interactions of E1253A, E1262A and E1262D mutants
with ATP have changed. One approach would require purifying the protein, and
measuring ATP binding and hydrolysis, and/or ADP release. Alternately, measurement of
ATP binding has commonly been measured by photolabeling with 8-azido-[γ-32P]ATP
91
under non-hydrolytic conditions. Similarly, “trapping” experiments using 8-azido-[α32
P]ATP at 37oC (which permits ATP hydrolysis and trapping of 8-azido-[α-32P]ADP in
the presence of sodium orthovanadate) could be carried out to determine if changes in
transport activities are associated with changes in ATP hydrolysis or ADP release (Koike
et al., 2004; Letourneau et al., 2007). However, given the differences in the structures of
azido-ATP and ATP, these experiments may have some limitations.
Suggested future studies of Arg1263 could involve the generation of the same
charge mutation (i.e. R1263K) to determine if the positive charge at this position is the
determining factor for maintaining MRP1 transport function. In addition, kinetic analyses
of LTC4 uptake of Arg1263 mutants would help determine whether its reduced LTC4
transport is caused by changes in apparent Km and/or Vmax, and furthermore, photolabeing
of these mutants by [3H]LTC4 would assist in supporting the findings from such kinetic
analyses.
In conclusion, a better understanding of how MRP1 structure relates to its
function is essential to understanding the basis of its diverse substrate specificity. Such
information could be used to aid in the rational design of selective inhibitors to counteract
MDR in tumours overexpressing MRP1. It could also be used to help in the design of
new drugs that could avoid MRP1-mediated efflux, so that under certain conditions,
exposure to therapeutics at tissues which are normally protected by MRP1 activity can be
prolonged. In addition, determination of MRP1 structure would be useful in predicting
drug-drug interactions with respect to the normal physiological roles of this transporter.
Thus, structure-function analyses of previously unexplored regions of MRP1 are of
current interest, since such studies could aid in elucidating the complex molecular
92
mechanisms of MRP1 activity. Since the cytoplasmic α-helical region COOH-proximal
to TM17 has not been previously characterized, the studies described in this thesis have
provided the first evidence of the functional importance of charged residues in this region.
These studies have also assisted in validating the accuracy of the current Sav1866-based
structural models of MRP1, and may one day contribute to the mapping of the multiple
substrate binding sites of MRP1.
93
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APPENDIX
A1.1 Uncharacterized MRP1 Mutants E1079A, R1263K, and E1263E Generated
and Related to this Study
Several Arg1263 mutants in pcDNA3.1 vectors were generated (see Chapter II, Section
2.4) (glycerol stocks of these vectors are stored at -80oC). These mutants (and the primers
used) include: R1263K (5’-GTG GCC GTG GAG AAG CTC AAG GAG-3’), R1263E
(5’-GTG GCC GTG GAG GAA CTC AAG GAG-3’), and E1079A (5’-GCT TCT CCA
AGG CGC TGG ACA CAG TG-3’).
A1.2 MRP1 Mutants R1202E, R1202E/E1204R, and R1202D/E1204K Generated
During the Course of this Master's Thesis in Collaboration with Marina Chan
Several Arg1202 mutants in pcDNA3.1 vectors were generated (see Chapter II, Section
2.4) (glycerol stocks of these vectors are stored at -80oC). These mutants (and the primers
used) include: R1202E (5’-GGC TGG CCG TGG AGC TGG AGT GTG-3’),
R1202E/E1204R (5’-CTG GCC GTG GAG CTG AGG TGT GTG GG-3’), and
R1202D/E1204K (sense DNA primer sequence of E1204R, to PCR ‘onto’ R1202D: 5’GGC CGT GGA T[CT TAA G]TG TGT GGG C-3’; AflII restriction site is in square
brackets).
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