Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition

Article abstract of DOI:10.1124/mol.107.043190

Reduced folate carrier (RFC) is the major membrane transporter for folates and antifolates in mammalian tissues. Recent studies used radioaffinity labeling with N-hydroxysuccinimide (NHS)-[³H]methotrexate (MTX) to localize substrate binding to

Full text of DOI:10.1124/mol.107.043190

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2008/01/09/mol.107.043190.DC1.html
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MOLECULAR PHARMACOLOGY
Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 73:1274–1281, 2008
Vol. 73, No. 4
43190/3317099
Printed in U.S.A.
Role of Lysine 411 in Substrate Carboxyl Group Binding
to the Human Reduced Folate Carrier, as Determined
□
S
by Site-Directed Mutagenesis and Affinity Inhibition
Yijun Deng, Zhanjun Hou, Lei Wang, Christina Cherian, Jianmei Wu, Aleem Gangjee,
and Larry H. Matherly
Graduate Program in Cancer Biology (Y.D., L.H.M.) and Department of Pharmacology (L.H.M.), Wayne State University School
of Medicine, Detroit, Michigan; Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit,
Michigan (Z.H., C.C., J.W., L.H.M.); and Division of Medicinal Chemistry, Graduate School of Pharmaceutical Science,
Duquesne University, Pittsburgh, Pennsylvania (L.W., A.G.)
Received November 2, 2007; accepted January 8, 2008
ABSTRACT
Reduced folate carrier (RFC) is the major membrane transporter gen- or methyl-substituted ␣-(2,3) or ␥-(4,5) carboxylates, or
for folates and antifolates in mammalian tissues. Recent studies substitutions of both ␣- and ␥-carboxylates (6–9) were used to
used radioaffinity labeling with N-hydroxysuccinimide (NHS)- inhibit [3H]MTX transport with RFC-null K562 cells expressing
[3H]methotrexate (MTX) to localize substrate binding to resi- wt and K411A RFCs. For wt and K411A RFCs, inhibitory po-
dues in transmembrane domain (TMD) 11 of human RFC. To tencies were in the order 4 Ͼ 5 Ͼ 1 Ͼ 3 Ͼ 2; 6 to 9 were poor
identify the modified residue(s), seven nucleophilic residues in inhibitors. Inhibitions decreased in the presence of physiologic
TMD11 were mutated to Val or Ala and mutant constructs anions. When NHS esters of 1, 2, and 4 were used to covalently
expressed in RFC-null HeLa cells. Only K411A RFC was not modify wt RFC, inhibitory potencies were in the order 2 Ͼ 1 Ͼ
inhibited by NHS-MTX. By radioaffinity labeling with NHS- 4; inhibition was abolished for K411A RFC. These results es-
[3H]MTX, wild-type (wt) RFC was labeled; for K411A RFC, ra- tablish that Lys411 participates in substrate binding via an ionic
diolabeling was abolished. When Lys411 was replaced with Ala, association with the substrate ␥-carboxylate; however, this is
Arg, Gln, Glu, Leu, and Met, only K411E RFC showed substantially not essential for transport. An unmodified ␣-carboxylate is re-
decreased transport. Nine classic diamino furo[2,3-d]pyrimidine quired for high-affinity substrate binding to RFC, whereas the
antifolates with unsubstituted ␣- and ␥-carboxylates (1), hydro- ␥-carboxyl is not essential.
Folates are important cofactors for transferring one-carbon molecules with ionized ␣- and ␥-carboxyl groups at physio-
units in biosynthetic steps leading to thymidylate, purine
nucleotides, and the amino acids serine and methionine
(Stokstad, 1990). Because mammalian cells cannot synthe-
size folates de novo, these cofactors must be derived from
exogenous dietary sources. Folates are hydrophilic anionic
logic pH that do not cross biological membranes by diffusion
alone. Accordingly, mammalian cells have evolved sophisti-
cated transport systems for facilitating folate uptake. Al-
though folate uptake can occur by folate receptors, organic
anion transporters, and a proton-coupled folate transporter
(Matherly and Goldman, 2003; Zhao and Goldman, 2007), the
best characterized folate transporter is the ubiquitously ex-
This study was supported by grants CA53535 (to L.H.M.) and CA125153 (to
A.G. and L.H.M.) from the National Cancer Institute, National Institutes of pressed reduced folate carrier (RFC) (SLC19A1) (Matherly et
Health.
al., 2007). Indeed, given its widespread tissue expression,
RFC is considered the major transport system for folates in
mammalian cells and tissues.
Membrane transport by RFC is also important for antitu-
mor activities of antifolates used for cancer chemotherapy
A.G. and L.H.M. contributed equally to this work.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.107.043190.
□S The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: RFC, reduced folate carrier; MTX, methotrexate; ZD9331, (2S)-2-{O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-
6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl)-butyric acid; PT523, N␣-(4-amino-4-deoxypteroyl)-N␦-hemiphthaloyl-L-ornithine;
TMD, transmembrane domain; hRFC, human reduced folate carrier; NHS, N-hydroxysuccinimide; ZD1694, N-(5-[N-(3,4-dihydro-2-methyl-4-
oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-L-glutamic acid; wt, wild type; HA, hemagglutinin; HSM, HEPES-sucrose-Mg2ϩ; DPBS,
Dulbecco’s phosphate-buffered saline; HBSS, Hanks’ balanced salt solution; ICI-198583, (S)-2-[(1-{4-[(2-methyl-4-oxo-3,4-dihydro-quinazolin-
6-ylmethyl)-prop-2-ynyl-amino]-phenyl}-methanoyl)-amino]-pentanedioic acid.
1274

Substrate Carboxyl Group Binding in hRFC Transport
1275
such as methotrexate (MTX), pemetrexed, and raltitrexed (Westerhof et al., 1995; Jansen, 1999), to date, no systematic
(Tomudex) (Jansen et al., 1999; Goldman and Zhao, 2002; study of the ␣- versus ␥-carboxylates in binding and trans-
Matherly et al., 2007). Losses of RFC function are common port by RFC has been reported.
mechanisms of antifolate resistance in in vitro and in vivo
Cationic amino acids (Arg, Lys) localized within the RFC
models (Sirotnak et al., 1981; Schuetz et al., 1988; Gong et TMD-spanning segments can be envisaged to directly partic-
al., 1997; Jansen et al., 1998; Roy et al., 1998; Zhao et al., ipate in binding of anionic (anti)folate substrates. Of partic-
1998a,b, 1999; Wong et al., 1999; Drori et al., 2000; Sadlish et ular interest are Arg373 in TMD10 and Lys411 in TMD11
al., 2000) and likely contribute to clinical resistance in pa- [numbering refers to human RFC (hRFC) sequence (Gen-
tients with osteosarcoma (Guo et al., 1999) and B-precursor Bank accession no. U19720)]. Both of these highly conserved
acute lymphoblastic leukemia who are treated with MTX (Ge amino acids were previously found to be important for RFC
et al., 2007). MTX has other clinical applications including transport (Sharina et al., 2001; Sadlish et al., 2002; Witt and
treatment of autoimmune diseases and psoriasis (Giannini et Matherly, 2002) and as likely candidates to participate in
al., 1992; Chla´dek et al., 1998).
binding associations with ionized ␣- and ␥-carboxyl groups in
The functional properties for RFC were first documented folate substrates. An unidentified nucleophilic amino acid in
nearly 40 years ago in murine leukemia cells (Goldman et al., TMD11 in hRFC was implicated as the major site of covalent
1968). However, it is only since the cloning of RFCs from modification by the activated carboxyl group(s) in N-hydroxy-
various species (Dixon et al., 1994; Williams et al., 1994; succinimide (NHS) [3H]MTX (Witt et al., 2004; Hou et al.,
Moscow et al., 1995; Prasad et al., 1995; Williams and Flint- 2005), an established affinity inhibitor of RFC (Henderson
off, 1995; Wong et al., 1995) and the application of molecular and Zevely, 1984; Matherly et al., 1991).
biology approaches to engineer RFC for biochemical studies
In this report, we directly explore the role of Lys411 in
that a detailed picture of the molecular structure of this TMD11 of hRFC in the binding and transport of anionic
physiologically important carrier has emerged, including its folate substrates and provide a structure-activity relation-
membrane topology, its N-glycosylation, and identification of ship of the substrate carboxylates for RFC transport. Our
functionally and structurally important domains and amino results establish that Lys411 participates in transport sub-
acids (for review, see Matherly et al., 2007). In contrast, there strate binding to hRFC and is the primary site for covalent
is a dearth of information regarding the structural require- modification by NHS-MTX. Through the use of a novel series
ments of RFC substrates. Because RFC is an anion trans- of furo[2,3-d]pyrimidine antifolates with substituted carboxyl
porter, the role of the terminal glutamate in transport is of groups on the terminal glutamate, we demonstrate that an
particular interest. Although modifications of the glutamate ionizable ␣-carboxyl group, but less so a ␥-carboxyl group, is a
␥-carboxyl group in RFC substrates were tolerated (e.g., va- critical substrate feature for high-affinity binding to hRFC. To
line), including those for the antifolates ZD9331 and PT523 our knowledge, this is the first report to systematically charac-
NH2
N
*
N
COOH
COOH
H
N
O
H2N
O
1
Compound
R1
COOH
CH3
R2
COOH
COOMe
COOH
COOEt
COOH
CH3
1
2a
2
R1
*
CH3
NH2
*
H2N
R2
R1
3a
3
H
*
N
N
R2
H
NH
O
H2N
*
O
4a
4
COOMe
COOH
COOMe
COOH
CH3
H2N
H2N
H2N
H2N
COOMe HCl
11
12
13
2a-5a
2-5
b
CH3
COOEt HCl
COOMe
NH2
N
*
a
HCl
HCl
5a
5
H
COOMe
*
N
14
H
O
H2N
COOH
6
CH3
10
R1
*
7
H
CH3
NH2
*
H2N
R2
R1
*
8
CH3
H
N
N
R2
*
NH
O
H2N
9
H
H
15
16
17
H2N
O
H2N
H2N
H2N
6-9
18
Fig. 1. Synthesis of furo[2,3-d]pyrimidine antifolates 1 to 9. A scheme is shown for the structures and synthesis of antifolates 1 to 9. Reaction
conditions are: a, N-methyl morpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, dimethylformamide, room temperature, 6 h; and b, 1 N NaOH, MeOH,
room temperature, 10 h and 1 N HCl. Compounds 13 and 17 have the same configuration as L-glutamate.

1276
Deng et al.
terize molecular features of substrate binding to RFC in light of cloned in pCDNA3 vector (Invitrogen) (Payton et al., 2007). Primers
for site-directed mutagenesis were designed according to the instruc-
tions for the QuickChange kit and are summarized in Supplemental
Table 1S. All mutations were confirmed by DNA sequencing at the
Wayne State University DNA sequencing core.
specific structural motifs in the (anti)folate molecule and par-
ticular conserved amino acids lining the membrane transloca-
tion pathway.
Western Analysis of Mutant hRFC Transfectants. Plasma
membranes were prepared by differential centrifugation (Matherly
et al., 1991). For standard Western blotting, membrane proteins
were electrophoresed on 7.5% polyacrylamide gels in the presence of
SDS (Laemmli, 1970) and electroblotted onto polyvinylidene difluo-
ride membranes (Pierce, Rockford, IL) (Matsudaira, 1989). hRFC
proteins were detected with HA-specific mouse antibody (Covance,
Berkeley, CA) and secondary IRDye 800-conjugated antibody (Rock-
land Immunochemicals, Gilbertsville, PA). Detection and densitom-
etry of the blots were performed with the Odyssey Imaging System
(LI-COR, Lincoln, NE).
Materials and Methods
Reagents. [3Ј,5Ј,7-3H]MTX (46.8 Ci/mmol) was purchased from
Moravek Biochemicals (Brea, CA). Unlabeled MTX was provided by
the Drug Development Branch, National Cancer Institute, National
Institutes of Health (Bethesda, MD). Both labeled and unlabeled
MTX were purified by high-performance liquid chromatography be-
fore use (Fry et al., 1982). The sources of the antifolate drugs were as
follows: raltitrexed and ZD9331 were obtained from AstraZeneca
Pharmaceuticals (Maccesfield, Cheshire, UK); pemetrexed was from
Eli Lilly & Co. (Indianapolis, IN); and PT523 was a gift of Dr. Andre
Rosowsky (Dana Farber Cancer Institute, Boston, MA). Synthetic
oligonucleotides were obtained from Invitrogen (Carlsbad, CA). Tis-
sue culture reagents and supplies were purchased from assorted
vendors with the exception of fetal bovine and supplemented calf
sera, which were purchased from Hyclone Technologies (Logan, UT).
Molecular biology reagents were from Promega (Madison, WI) or
Invitrogen.
Synthesis of Furo[2,3-d]Pyrimidine Antifolates 1 to 9. Com-
pound 10, 4-{1-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]propyl}-
benzoic acid, was obtained as described previously (Gangjee et al.,
2002). For compounds 2 to 5, 10 was coupled with the appropriate
commercially available, modified, glutamate ester analogs with N-
methylmorpholine and 2-chloro-4,6-dimethoxy-1,3,5-triazine in dim-
ethylformamide at room temperature for 6 h to afford the desired
esters in approximately 60% yield (see Fig. 1). Saponification with
aqueous sodium hydroxide at room temperature followed by acidifi-
cation to pH 4 in an ice bath afforded 2 to 5 in approximately 95%
yield. For compounds 6 to 9, 10 was similarly coupled with the
appropriate commercially available amines to give the desired prod-
ucts. A reaction scheme for the synthesis of analogs 1 to 9 is shown
in Fig. 1. Detailed syntheses are provided in the Supplemental
Materials and Methods.
Membrane Transport Assays. Uptake of [3H]MTX (0.5 ␮M) in
transiently transfected R5 HeLa cells was measured over 2 min at
37°C in 60-mm dishes in an “anion-free” HEPES-sucrose-Mg2ϩ
(HSM) buffer (20 mM HEPES, 235 mM sucrose, pH adjusted to 7.14
with MgO). Uptake of [3H]MTX was quenched with ice-cold Dulbec-
co’s phosphate-buffered saline (DPBS). Cells were washed with ice-
cold DPBS (three times) and solubilized with 0.5 N NaOH. [3H]MTX
(1 ␮M) uptake into stably transfected K500E cells was measured
over 180 s (wt and Lys411 mutant) in both HSM buffer and physio-
logic Hanks’ balanced salt solution (HBSS) in a shaking water bath
at 37°C, as described previously (Wong et al., 1997). For both cell line
models, levels of intracellular radioactivity were expressed as pico-
moles per milligram of protein, calculated from direct measurements
of radioactivity and protein contents of cell homogenates. Protein
assays were based on the method of Lowry et al. (1951). For the
stable transfected K500E cells, kinetic constants (Kt, Vmax) were
calculated from Lineweaver-Burk plots for [3H]MTX, and Ki values
for assorted antifolate substrates were determined from Dixon plots
with [3H]MTX (1 ␮M).
3
No treatment
Cell Culture. Transport-defective MTX-resistant HeLa cells, des-
ignated R5 (Zhao et al., 2004), were a generous gift of Dr. I. David
Goldman (Bronx, New York). R5 cells were maintained in RPMI
1640 and supplemented with 10% fetal bovine serum, 2 mM L-
glutamine, penicillin (100 units/ml), and streptomycin (100 ␮g/ml) in
a humidified atmosphere at 37°C in the presence of 5% CO2. Tran-
sient transfections of wild-type (wt) and mutant hRFC constructs
(see below) were performed with Lipofectamine Plus reagent (In-
vitrogen), as described previously (Hou et al., 2005, 2006). Cultures
were split 24 h after transfection and assayed for transport and
expression on Western blots after an additional 24 h.
hRFC
NHS-Mtx
2
*
*
*
1
*
*
*
*
0
The MTX transport-deficient K562 subline, designated K500E,
was selected from wt K562 cells (American Type Culture Collection,
Manassas, VA) and maintained in complete RPMI 1640 medium
containing 10% supplemented calf serum, 2 mM L-glutamine, 100
U/ml penicillin, 100 ␮g/ml streptomycin, and 0.5 ␮M MTX in a
humidified atmosphere at 37°C in the presence of 5% CO2 (Wong et
al., 1997). Mutant and wt hRFC constructs (see below) were trans-
fected into K500E cells by electroporation (155 V, 1000-␮F capaci-
tance). After 24 h, cells were treated with G-418 (Geneticin; 1 mg/
ml), and stable clones were selected by cloning in soft agar in the
presence of 1 mg/ml G-418 (Wong et al., 1997). Transfected K500E
cultures were maintained in complete RPMI 1640 medium with
1 mg/ml G-418.
Fig. 2. NHS-MTX inhibition of nucleophilic amino acids in TMD11 of
hRFC. Seven nucleophilic amino acids in TMD11 (Cys396, Asn403,
Thr404, Thr408, Lys411, Thr412, Thr415) were mutated to non-nucleo-
philic amino acids (e.g., Ala or Val), and the wt and mutant hRFC
constructs were transiently transfected into R5 HeLa cells for comparison
with the vector control (labeled “Control”). hRFC proteins were assayed
on Western blots with 2.5 ␮g of membrane proteins and with HA-specific
mouse antibody and secondary IRDye 800-conjugated antibody (inset).
Detection used the Odyssey Imaging System. In the main panel, the R5
transfectants were treated with NHS-MTX (5 ␮M) and then assayed for
transport of [3H]MTX (0.5 ␮M) over 2 min. In results from five separate
experiments, statistically significant inhibitions (p Ͻ 0.05 by Wilcoxon
test; asterisks) resulting from NHS-MTX treatments compared with un-
treated samples were measured for wt and TMD nucleophilic substitu-
tions, which ranged from 24% (for Thr412) to 60% (for Thr415). Only the
K411A hRFC mutant was completely inert to the NHS-MTX treatment.
In the figure, single-letter abbreviations for the amino acid replacements
at Lys411 are used. WT, wild type.
Site-Directed Mutagenesis of hRFC. hRFC mutants were gen-
erated by site-directed mutagenesis using the QuickChange kit
(Stratagene, La Jolla, CA) and a wt hRFC construct with a 5Ј-
untranslated region from positions Ϫ1 to Ϫ49, the full-length hRFC
open reading frame, and a hemagglutinin (HA) epitope at Gln587,

Substrate Carboxyl Group Binding in hRFC Transport
1277
Affinity Labeling of hRFC with NHS-MTX Ester. The prepa- 1992). Previous studies from our laboratory used NHS-
[3H]MTX with hRFC TMD1–6 and TMD7–12 half-molecules
ration of unlabeled and radiolabeled NHS-MTX was performed ex-
to localize covalent binding of [3H]MTX (via the substrate
actly as described previously (Matherly et al., 1991; Witt et al.,
2004). For treatments of R5 transfectants, NHS-MTX in 20 ␮l of dry
DMSO was added to 60-mm dishes of R5 cells in 2 ml of HSM buffer
al., 2005). Because TMD11 (but not TMD12) is directly in-
at room temperature for 5 min. For nonradioactive NHS-MTX, the
carboxyl groups) to TMDs 11 and 12 (Witt et al., 2004; Hou et
volved in substrate binding of hRFC substrates (Hou et al.,
final concentration was 5 ␮M. After treatment with NHS-MTX, cells
2005), we reasoned that one or more of the seven nucleophilic
were washed three times with DPBS at 0°C and assayed for
[3H]MTX (0.5 ␮M) transport (see above).
amino acids in TMD11 must be a direct target for covalent
modification by the NHS-MTX activated ester and, by exten-
sion, inhibition of transport activity.
For radioaffinity labeling experiments, NHS-[3H]MTX was pre-
pared at a radiospecific activity of 46.8 Ci/mmol; the final concentra-
tion of NHS-[3H]MTX with the cells was 700 nM. After radioaffinity
labeling, plasma membranes were prepared by differential centrifu-
gation. Membrane pellets were solubilized in 1% SDS and fraction-
ated on 1.5-mm 7.5% polyacrylamide gels with SDS, the gels were
sliced into 2-mm segments, and the pieces were suspended into 1 ml
of Soluene-350 (PerkinElmer Life and Analytical Sciences, Waltham,
MA) overnight at room temperature. Five milliliters of Ready-value
scintillation cocktail (Beckman Coulter, Fullerton, CA) were added,
and radioactivity was detected on a model 6500 Beckman liquid
scintillation counter.
Accordingly, we mutated each of the seven nucleophilic
amino acids in TMD11 (Cys396, Asn403, Thr404, Thr408,
Lys411, Thr412, Thr415) to non-nucleophilic amino acids
(Ala or Val). Mutant and wt hRFC proteins were expressed in
hRFC-null R5 HeLa cells, and all were found to be capable of
transporting MTX (0.5 ␮M) within an approximately 3-fold
range of activities (Fig. 2). When the R5 transfectants were
treated with NHS-MTX (5 ␮M) then assayed for [3H]MTX
transport, for six of seven hRFC mutants and wt hRFC,
transport was inhibited. Statistically significant inhibitions
resulting from NHS-MTX were measured that ranged from
24% (for Thr412) to 60% (for Thr415). Only the K411A hRFC
mutant was completely and reproducibly inert to NHS-MTX
treatment.
Because loss of RFC activity by NHS-MTX treatment is the
result of a covalent modification of the carrier (Henderson
and Zevely, 1984; Schuetz et al., 1988; Matherly et al., 1991;
Witt et al., 2004), the lack of a transport effect on the K411A
mutant suggested that Lys411 is a likely target for electro-
philic attack by the activated NHS-MTX ester. To directly
test this possibility, we transiently transfected R5 cells with
wt and K411A hRFC constructs, then treated the cells with
NHS-[3H]MTX (700 nM) so as to radiolabel the carrier.
Plasma membranes were prepared and detergent-solubi-
lized, and the solubilized radiolabeled proteins were fraction-
NHS esters of furo[2,3-d]pyrimidine antifolate analogs were pre-
pared from the parent compounds exactly as for MTX. For treat-
ments of the stable transfected K500E cells with the NHS-furo[2,3-
d]pyrimidine antifolates, cells were treated in 2 ml of HSM buffer, as
described above for the R5 cells, albeit in suspension in a shaking
water bath at room temperature. Cells were washed with ice-cold
DPBS and assayed for MTX (1 ␮M) transport in HBSS.
Results
Identification of Lys411 as the Primary Site of Cova-
lent Labeling by NHS-MTX. NHS-MTX is an established
affinity inhibitor of hRFC (Henderson and Zevely, 1984;
Matherly et al., 1991). NHS esterification activates the car-
boxyl groups of MTX and related compounds for electrophilic
reaction with biological nucleophiles. NHS-[3H]MTX and
-aminopterin, have been extensively used for covalently la-
beling the carrier in cultured cells (Henderson and Zevely, ated on a 7.5% polyacrylamide gel for direct counting. Similar to
1984; Schuetz et al., 1988; Matherly et al., 1991; Yang et al., previous reports (Matherly et al., 1991; Witt et al., 2004), for wt
kDa
250 148
98
64
50
DF
36
6000
5000
4000
3000
2000
1000
0
wild type
Fig. 3. Radioaffinity labeling of wt hRFC and
K411A hRFC with NHS-[3H]MTX. R5 cells were
transfected with wt and K411A hRFC con-
structs. Cells were treated with NHS-[3H]MTX
(700 nM), as described under Materials and
Methods. Membranes were prepared, and pro-
teins (150 ␮g) were fractionated on 7.5% poly-
acrylamide gels in the presence of SDS. The gels
were sliced into 2-mm segments (labeled “Frac-
tions” on the abscissa), and the radioactivity was
extracted and directly counted. The positions of
molecular mass standards (in kilodaltons) are
shown. hRFC migrates on SDS gels as a broadly
banding species centered at 80 to 85 kDa as
previously reported (Matherly et al., 1991; Witt
et al., 2004). DF, dye front.
Lys411Ala
0
10
20
30
40
50
60
Fractions

1278
Deng et al.
hRFC expressed in R5 cells, incorporation of radioactivity from stituted ␣- (2, 3) or ␥- (4, 5) carboxyl groups and with substi-
NHS-[3H]MTX involved a broadly migrating hRFC protein cen- tutions of both ␣- and ␥-carboxyl groups (6–9) (Fig. 1). We
tered at ϳ80 to 85 kDa (Fig. 3). Substitution of Lys411 by Ala initially used these analogs as reversible inhibitors (at 10
dramatically and nearly completely abolished incorporation of ␮M) of radioactive MTX (1 ␮M) uptake with hRFC-null K562
radioactivity into hRFC, clearly establishing Lys411 as the (K500E) cells stably transfected with wt and K411A hRFC.
primary target for covalent modification by NHS-MTX and Kinetic constants (Vmax and Kt) for MTX with wt and K411A
implicating Lys411 as involved in the binding of (anti)folate hRFC are summarized in Table 1, and a Western blot of wt
substrates via ionic associations with the ␣- and/or ␥-carboxyl and K411A hRFC proteins in the transfected cells is shown in
groups of the terminal glutamate.
Supplemental Fig. 1S.
Effects of Conservative and Nonconservative Re-
In physiologic HBSS buffer, the ␥-substituted 4 and 5
placements of Lys411 in TMD11 of hRFC. The results in analogs showed potent inhibitions of MTX transport for both
Fig. 3 clearly identify Lys411 as the primary modification wt and K411A hRFC (Fig. 5, A and B), whereas the parental
site for NHS-[3H]MTX and, by inference, as important for drug, 1, and ␣-substituted 2 and 3 analogs were weaker
substrate carboxyl group binding to hRFC. It is interesting inhibitors. hRFC transport activity was stimulated (up to
that both conservative and nonconservative substitutions at 5-fold) in the absence of competing anions (i.e., in anion-free
Lys411, including Ala, Glu, Leu, Met, Gln, and Arg, were well HSM buffer) (data not shown), presumably reflecting de-
tolerated when mutant hRFC constructs were transiently creased competition for anionic substrate binding by anions
expressed in hRFC-null R5 HeLa cells, along with wt hRFC, (e.g., ClϪ) in HSM buffer. Consistent with this, the inhibitory
and assayed for MTX transport (Fig. 4). Indeed, when trans- effects on [3H]MTX uptake by all of the anionic antifolates
port results were normalized to hRFC protein on Western with one or two carboxyl groups (compounds 1–5) were en-
blots (lower panel), only K411E showed a substantial de- hanced in HSM buffer (Fig. 5, C and D). Although the in-
crease in drug uptake (ϳ50% of wt). Similar results were crease was greatest for 1, compounds 4 and 5 remained the
reported elsewhere for hRFCs with a smaller number of most potent inhibitors. In contrast, analogs with neither ␣-
Lys411 mutants (Witt and Matherly, 2002; Hou et al., 2005). nor ␥-carboxyl groups (6–9) were exceedingly poor inhibitors
Further Characterization of the Role of Lys411 in of MTX uptake in both HBSS and HSM buffers. Ki values for
Substrate Binding with Diamino Furo[2,3-d]Pyrimi- the antifolates 4 and 5 with wt and K411A hRFCs (in phys-
dine Antifolates with Substituted Carboxyl Groups. iologic buffer) are summarized in Table 1. Results are con-
Gangjee et al. (2002) previously reported the synthesis and sistent with higher affinity binding for antifolates 4 and 5
biological activities of a dihydrofolate reductase inhibitor and than for pemetrexed and raltitrexed and similar binding
RFC substrate, N- [4-[1-ethyl-2-(2,4-diaminofuro[2,3-d]pyri- affinities to those for the classic hRFC high-affinity sub-
midin-5-yl)ethyl]-benzoyl]L-glutamic acid (designated 1; Fig. strates PT523 and ZD9331.
1), as a growth inhibitor of CCRF-CEM leukemia cells in
These results indicate that although both substrate ␣- and
culture. To further characterize the relative importance of ␥-carboxyl groups participate in substrate binding to hRFC,
the ␣- and ␥-carboxyl groups of (anti)folate substrates for it is the binding of the ␣-carboxyl group that predominates
binding with Lys411 of hRFC, we synthesized a series of and is indeed essential for high-affinity binding. From the
furo[2,3-d]pyrimidine analogs with methyl- or hydrogen-sub- results with the 1 versus 4/5 antifolates in HBSS, it appears
that the ␥-carboxyl can negatively affect ␣-carboxyl group
binding to the carrier. Finally, the presence of a cationic
amino acid at position 411 is clearly not necessary for reversible
230
binding of the furo[2,3-d]pyrimidine antifolates 1 to 5 to hRFC.
Affinity Inhibition of wt and K411A hRFC by NHS
Esters of Diamino Furo[2,3-d]Pyrimidine Antifolates
with Substituted Carboxyl Groups. To further explore
the associations between the ␣- and ␥-carboxyl groups of
(anti)folate substrates and Lys411, compound 1 and its ␣-
and ␥-methyl-substituted congeners, 2, 4, and 6, were treated
Lys411
210
100
50
0
TABLE 1
Ki values for furo͓2,3-d͔pyrimidine antifolates 4 and 5 and classical
antifolates with wt and K411A hRFCs
WT
A
E
L
M
Q
R
Kinetic constants were determined from Lineweaver-Burk and Dixon plots for the wt
and K411A hRFC transfectants using ͓3H͔MTX. The data shown are the mean
values from three experiments Ϯ S.E.M.
hRFC
Parameter
Antifolate
Wild-Type hRFC
K411A hRFC
Fig. 4. Site-directed mutagenesis of Lys411 and expression in R5 HeLa
cells. Transport activity and Western blot data are shown for position 411
substitutions, as described in the text. Lower panel, results for a Western
blot of proteins (2.5 ␮g) solubilized from membrane preparations from R5
HeLa cells transiently transfected with wt and Lys411 mutant hRFC
constructs. hRFC proteins were detected with HA-specific mouse anti-
body and secondary IRDye 800-conjugated antibody. Detection and den-
sitometry of the blots were performed with the Odyssey Imaging System.
Upper panel, uptake data for [3H]MTX (0.5 ␮M) over 2 min at 37°C,
normalized to hRFC protein levels from the Westerns. Results presented
are representative of three experiments. Single-letter abbreviations for
the amino acid replacements at Lys411 are used. WT, wild type.
␮M
Kt
Vmax
Ki
MTX
MTX
1.23 Ϯ 0.23
4.76 Ϯ 0.87a
1.5 Ϯ 0.2
2.5 Ϯ 0.2
9.9 Ϯ 0.7
5.3 Ϯ 0.4
2.9 Ϯ 0.2
2.4 Ϯ 0.2
1.58 Ϯ 0.17
2.76 Ϯ 0.32a
3.2 Ϯ 0.2
4
5
Ki
4.4 Ϯ 0.6
Ki
Pemetrexed
Tomudex
ZD9331
PT523
15.1 Ϯ 1.5
11.2 Ϯ 0.2
5.4 Ϯ 0.5
Ki
Ki
Ki
5.0 Ϯ 0.6
a Picomoles per minute per milligram of protein.

Substrate Carboxyl Group Binding in hRFC Transport
1279
with NHS, using methods identical to those for preparing affinity inhibition of MTX transport by 1 to 6 was largely
NHS-MTX. The activated NHS-antifolate esters (5 ␮M) were abolished.
added to the K500E transfectants expressing wt and K411A
Thus, for NHS antifolate activation and covalent modifica-
hRFC (in HSM buffer) to determine effects on MTX transport tion of the carrier, the ␥-carboxyl is clearly preferred, in
activity resulting from covalent modification of the carrier, contrast to the results for reversible binding in which the
analogous to the experiment in Fig. 2 with NHS-MTX and furo[2,3-d]pyrimidine antifolates bearing ionizable ␣-car-
transfected R5 cells. In striking contrast to the results for boxyl groups were more potent inhibitors than those with
reversible inhibition of transport by the unmodified furo[2,3- ␥-carboxyl groups alone or with both ␣- and ␥-carboxyl
d]pyrimidine antifolates (Fig. 5), for the NHS-activated ana- groups. Because affinity labeling was nearly completely abol-
logs, the order of inhibition with wt hRFC was 2 Ͼ 1 Ͼ 4 with ished for K411A for both NHS-MTX (see above) and the
potencies ranging from 70% inhibition down to 30% inhibi- NHS-esters of the furo[2,3-d]pyrimidine antifolates, the
tion (Fig. 6). Not surprisingly, there was no inhibition by ␥-carboxyl groups of transport substrates must associate
analog 6, which has no carboxyl groups. For K411A hRFC, with Lys411 in TMD11 of hRFC.
Wild type - HBSS
Lys411Ala - HBSS
B
100
A
100
Fig. 5. Inhibition of MTX trans-
port in K500E transfectants
expressing wt and K411A hR-
FCs with diamino furo[2,3-d]-
pyrimidine antifolates
1 to 9.
hRFC-null K500E cells were sta-
bly transfected with wt hRFC
and K411A hRFC, as described
under Materials and Methods.
Clones were isolated, expanded,
and assayed for hRFC levels on
Western blots (Supplemental
Fig. 1S), and kinetic constants
were determined for MTX (Ta-
ble 1). In the experiment shown,
wt and K411A were assayed for
[3H]MTX uptake (1 ␮M) in the
presence of the ␣- and ␥-carboxyl-
substituted diamino furo[2,3-
d]pyrimidine antifolates 1 to 9
(10 ␮M), as described in the
text. The structures for com-
pounds 1 to 9 are shown in Fig.
1. The experiments shown in
A and B used HBSS for trans-
port, whereas the experiments
shown in C and D used anion-
free HSM buffer. Results are
shown as mean values Ϯ S.E.M.
for three experiments. NA, no
additions.
50
0
50
0
NA
1
2
3
4
5
6
7
8
9
NA
1
2
3
4
5
6
7
8
9
C
D
Wild type - HSM
Lys411Ala - HSM
100
50
0
100
50
0
NA
1
2
3
4
5
6
7
8
9
NA
1
2
3
4
5
6
7
8
9
Lys411Ala hRFC
Wild type hRFC
110
100
110
100
50
0
50
NA
1
2
4
6
NA
1
2
4
6
Fig. 6. Inhibition of MTX transport in stable K500E-transfected cells expressing wt and K411A hRFC with activated NHS esters of antifolates 1, 2,
and 4. Analogs 1, 2, and 4 with ␣- and/or ␥-carboxyl groups (structures are shown in Fig. 1) and 6 with both ␣- and/or ␥-carboxyl groups substituted
with methyls were treated with NHS, using methods identical to those for preparing NHS-MTX. The activated NHS-antifolate esters (5 ␮M final
concentrations) were added to the K500E transfectants expressing wt and K411A hRFC for 5 min; the cells were washed and assayed for the effects
on [3H]MTX (1 ␮M) uptake over 180 s. Data are expressed as relative [3H]MTX uptakes (in picomoles per milligram of protein; mean values Ϯ S.E.M.;
n ϭ 3) after treatment with the NHS esters of the furo[2,3-d]pyrimidine antifolates, and are compared with the level in untreated cells. NA, no
additions.

1280
Deng et al.
tetrazole in ZD9331 is an isosteric anionic replacement for
the ␥-carboxylate.
Discussion
Folates are composed of distinct structural motifs includ-
ing pteridine, p-aminobenzoate, and glutamate. The gluta-
mate moiety is of particular importance in that its ␣- and
␥-carboxyl groups are ionized at physiologic pH, thus limiting
diffusion of folates and classic antifolates across biological
membranes. Because RFC is a transporter of organic anions,
in this study, we focused on the mechanistic role of the
substrate carboxyl groups in transport by hRFC. Analysis of
membrane topology models and sequence homologies for
RFCs from assorted species identified the highly conserved
cationic residues, Arg373 in TMD10 and Lys411 in TMD11,
as possibly functionally important (Matherly et al., 2007). By
site-directed mutagenesis, both these residues were previ-
ously implicated as important to RFC transport (Sharina et
al., 2001; Sadlish et al., 2002; Witt and Matherly, 2002) and
as likely candidates to participate in binding associations
with ionized ␣- and ␥-carboxyl groups in folate substrates.
This article further focuses on Lys411 and provides impor-
tant new insights into the relationship between antifolate ␣-
and ␥-carboxyl groups and this residue in TMD11, identified
as an important substrate binding domain and component of
the transmembrane translocation pathway in hRFC for an-
ionic folate and antifolate substrates (Hou et al., 2005). As
previously implied (Hou et al., 2006; Matherly et al., 2007),
the present results establish that Lys411 lies in the proxim-
ity of the aqueous substrate binding pocket in hRFC, where
it is subject to electrophilic attack by NHS-activated MTX
ester and can participate in an interaction with (anti)folate
substrate, primarily through an ionic association with the
␥-carboxyl group. Remarkably, this interaction is apparently
not essential for transport function because the ␥-carboxyl
group is not only expendable, but indeed its replacement by
an uncharged hydrogen or a methyl group in a series of
furo[2,3-d]pyrimidine antifolates actually enhances high-af-
finity reversible binding of substrate to the carrier, as long as
an ionizable ␣-carboxyl group is intact. Furthermore, Lys411
can be replaced by any of a number of amino acids of varying
bulk and charge with relatively nominal effects on overall
transport activity. From the apparently critical role of a
cationic amino acid at position 373 (Sharina et al., 2001;
Sadlish et al., 2002; Hou et al., 2006), we suggest that sub-
strate binding involves an ionic association between the
␣-carboxyl group of (anti)folate substrates and Arg373 in
TMD10 of hRFC. Because substrate binding is partially pre-
served for antifolate analogs 2 and 3 with blocked ␣- and
ionizable ␥-carboxylates, we propose that in the absence of
the ␣-carboxylate, the ␥-carboxylate can adopt a folded con-
formation so as to mimic the ␣-carboxylate.
Future studies will continue to focus on identification of
functionally important amino acids in hRFC and key sub-
strate-specific determinants of binding and translocation as
important steps to understanding the mechanism of folate
transport. Indeed, molecular insights from RFC structure-
function studies should foster the design of new antifolate
inhibitors that rely on RFC for cellular entry, or with sub-
stantially enhanced transport by other folate transporters
over RFC, and the development of strategies for biochemi-
cally modulating the carrier that could be therapeutically
exploited in the context of nutritional interventions or anti-
folate chemotherapy.
Acknowledgments
We thank I. David Goldman (Albert Einstein School of Medicine)
for the generous gift of hRFC-null R5 HeLa cells.
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Address correspondence to: Dr. Larry H. Matherly, Developmental Ther-
apeutics Program, Barbara Ann Karmanos Cancer Institute, 110 E. Warren
Avenue, Detroit, MI 48201. E-mail: matherly@kci.wayne.edu