(Received for publication, January 18, 1995; and in revised form, April 7, 1995)
From the
This report describes the isolation, nucleotide sequencing, and
functional expression of human cDNAs that restore reduced folate
carrier activity in transport-defective cells. Based on homology to a
partial murine cDNA probe, two functional cDNAs were isolated from a
The expression of both KS43 and
KS32 in methotrexate transport-defective Chinese hamster ovary cells
restored methotrexate sensitivity and transport. Certain transport
characteristics of the transfected cells resembled both the wild type
human (K562) and hamster ``classical'' reduced folate
carriers, suggesting the expression of a hybrid system. For instance,
based on K
Methotrexate (Mtx)
In order to better understand the
underlying cellular and molecular mechanisms associated with defective
membrane transport, a number of studies have focused on the
identification and molecular cloning of putative Mtx/reduced folate
transport proteins. Matherly et al.(14) used
radioaffinity labeling with N-hydroxysuccinimide-[
The
present study describes the isolation and expression of human cDNAs
which encode a function involved in the membrane transport of Mtx and
reduced folates. The cDNAs were isolated from a cDNA library prepared
from Mtx transport up-regulated K562.4CF cells, based on homology to
the mouse cDNA. Mtx transport-defective CHO cells transfected with the
human cDNAs regain their Mtx sensitivities and transport activities.
The transport characteristics of the transfected cells resemble both
the wild type human and hamster classical RFCs, suggesting the
expression of a hybrid transport system. In addition, the cDNA encoded
proteins can be radiolabeled with
APA-[
CHO wild
type Pro
For
cytotoxicity determinations, cells were cultured in RPMI 1640
supplemented with dialyzed fetal bovine serum and antibiotics, as
described above, in 24-well dishes at 20,000 (K562) or 10,000 (CHO)
cells/well. After 96 h of continuous exposure to various concentrations
of Mtx and 1843U89, cell viabilities were determined by direct
microscopic counting with a hemacytometer, either directly (K562) or
following trypsinization (CHO). IC
A
Figure 1:
Sequencing
strategy, restriction map, nucleotide sequence, and deduced amino acid
sequence of the human RFC cDNAs. A, a restriction map for the
composite sequence of KS32 and KS43 is shown. The solid horizontal
bars represent the open reading frame. The shaded horizontal
bars represent the heterogeneous 5`-untranslated sequences. The break in the KS32 line indicates deletion in the sequence with
respect to KS43. The arrows indicate the direction and extent
of nucleotide sequence obtained from each primer as described. B, the complete nucleotide sequence (upper
line) and the deduced amino acid sequence (lower line)
for the KS43 cDNA clone are shown. The numbering system is relative to
the putative start codon (29) at position 1. The termination
codon is indicated by a plus (+) sign. The putative
polyadenylation signal is indicated by double underlined bases. Single underlined bases represent the deleted
sequence in clone KS32. The 14 bold faced and italicized bases, starting at position 2194, are included in the KS32
putative coding region.
Figure 2:
The heterogenous 5` sequences of hRFC
cDNAs. The heterogenous untranslated 5` sequences of clones KS6, KS32
and KS43 are shown. The numbering indicates the nucleotide position
relative to the putative translational start site (assigned as position
1). The underlined sequences indicate where the identical
5`-untranslated regions for all three clones
commence.
Figure 3:
Amino acid sequence alignment for the
human, mouse, and hamster RFCs. The predicted amino acid sequence of
human RFC from KS43 is compared to the predicted amino acid sequences
from the mouse (20) and hamster (21) cDNAs. Gaps,
indicated by a dash(- - -), are introduced for the alignment.
Where the mouse or hamster amino acid is conserved relative to the
human sequence, it is indicated by a period.
Figure 4:
Northern blot analysis of RNAs prepared
from K562 sublines. Upper panel, Total RNA (20 µg) was
electrophoresed, blotted onto a nylon membrane, and hybridized with the
[
Both Mtx RII Oua
Figure 5:
Initial uptake rates of
[
The
restored drug sensitivities and [
Figure 6:
Northern blot analysis of CHO and K562
RNAs. Upper panel, total RNA (20 µg) was electrophoresed,
blotted onto a nylon membrane, and hybridized with the
[
Figure 7:
Photoaffinity labeling of hRFC with
APA-[
The present study describes the isolation, nucleotide
sequence analysis, and functional expression of human cDNAs which
restore Mtx sensitivity and membrane transport in Mtx-resistant Mtx RII
Oua
The nucleotide
sequence and predicted protein structure of the encoded hRFC showed
close homology to the recently described mouse (20) and hamster (21) cDNAs. The larger size of the human transcript (3.1 versus 2.5 kb) appears to reflect not only a larger ORF but
also differences in both 5`- and 3`-untranslated regions. The
prediction of multiple hydrophobic domains with membrane spanning
potential and the presence of a putative N-glycosylation site
with high surface probability substantiates our earlier studies of a
glycosylated 92-kDa integral membrane carrier protein in transport
up-regulated K562 cells which can be affinity labeled with
NHS-[
The restoration of Mtx
sensitivities and capacities for Mtx transport by both the full-length
and the 3`-truncated human cDNAs suggests that the deleted 3`-ORF
sequence in KS32 has little direct role on membrane insertion. The
hydrophilic peptide encoded by the deleted ORF in KS32, nonetheless,
appears to influence the binding of certain transport substrates (i.e. folic acid, leucovorin) and overall transport
efficiency.
The extensive heterogeneity in the 5`-non-coding
sequences of the hRFC cDNAs may reflect the alternative splicing of
transcripts transcribed from multiple promoters or transcription
initiation sites(33) . The 3`-truncated KS32 clone may have a
similar origin or represent a cloning artifact. Of course, the
detection of a major 3.1-kb mRNA in K562 cells and sublines with widely
disparate capacities for Mtx transport implies that only the hRFC
encoded by the longer KS43 transcript is significantly expressed in
these lines. Furthermore, only a single labeled protein was detected in
K562 cells treated with APA-[
Of
particular interest was the finding that the restored
[
Our earlier
studies suggested a causal role for a GP-Mtx, a highly glycosylated
membrane component, in Mtx transport(15) . GP-Mtx was
originally believed to be a Mtx carrier since it could be radioaffinity
labeled with NHS-[
Whereas the identity of
GP-Mtx is presently uncertain, it is interesting that most of the
immunoreactive GP-Mtx protein from K562 plasma membranes partitions
into the detergent-poor phase in Triton X-114 phase separation
experiments,
In
conclusion, our results show that the hRFC cDNAs are capable of
restoring Mtx sensitivity and carrier-mediated Mtx membrane transport
in Mtx transport defective CHO cells, apparently by expressing the
``classical'' RFC. Surface hRFC proteins were specifically
labeled with APA-[
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U19720[GenBank® Link].
We thank Daryel Taliaferro for secretarial assistance
in preparing this manuscript. We also thank Dr. Wayne Flintoff for
providing the wild type (Pro
Note Added
in Proof-Since the submission of this article, two other
homologous human cDNAs have been reported (Refs. 36, 37). In both of
these instances, numerous differences at the nucleotide level within
the open reading frames were noted compared to the full-length hRFC
cDNA sequence. These differences were represented by rearrangements,
substitutions, or deletions. Due to these differences, the amino acid
sequences encoded by these cDNAs are 90-95% homologous to that
being reported here.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
gt11 library prepared from methotrexate transport up-regulated
K562 cells (K562.4CF). A 2.8-kilobase (kb) clone, KS43, contained a
1776-base pair open reading frame. The 2.5-kb clone, KS32, contained an
internal deletion (626 base pairs) resulting a shortened open reading
frame and 3`-untranslated region. KS43 and KS32 encoded proteins with
multiple hydrophobic domains, one consensus N-glycosylation
site, and predicted molecular masses of 65 and 58 kDa, respectively.
The deduced amino acid sequence of KS43 is 79% and 80% homologous to
the mouse and hamster sequences, respectively (Dixon, K. H., Lanpher,
B. C., Chiu, J., Kelley, K., and Cowan, K. H.(1994) J. Biol. Chem. 269, 17-20; Williams, F. M. R., Murray, R. C., Underhill, T.
M., and Flintoff, W. F.(1994) J. Biol. Chem. 269,
5810-5816). Northern blots identified one primary transcript at
3.1 kb in parental K562, K562.4CF, and transport-impaired K500E cells;
transcript levels varied by 7-fold.
values, up to a 4-fold
increased affinity for 1843U89 over wild type hamster cells (typical of
human cells), and a 19-fold increased affinity for methotrexate over
K562 cells (typical of hamster cells) was observed. Further, a
photoaffinity probe with high specificity for the reduced folate
carrier labeled 94-kDa proteins in K562 cells and the transfectant
containing the full-length KS43, and a 85-kDa protein in the
transfectant containing the 3`-truncated KS32. No specifically labeled
proteins were detected in wild type or mock-transfected hamster cells.
Collectively, our results suggest that the KS43/KS32 cDNAs encode the
human reduced folate carrier; however, additional modulatory/regulatory
factors may be required to manifest the full spectrum of transport
substrate activities typical of this system.
(
)as well as some
newer and more potent antifolates are actively transported
intracellularly via a carrier-mediated uptake
system(1, 2, 3, 4) . In experimental
and clinical neoplastic cells, impaired membrane transport has been
recognized as one of the major mechanisms of Mtx resistance (4, 5, 6) . The carrier system is
characterized by its saturability and marked temperature dependence, as
well as its high affinity for Mtx and reduced
folates(7, 8) . By contrast, this system exhibits a
low affinity for folic acid and, hence, transports this compound
poorly(7, 8) . In addition to this
``classical'' Mtx/reduced folate carrier (RFC), a low
affinity/high capacity transport system for folic acid has been
reported(9) . Other membrane folate ``binding
proteins'' have also been described in various cells and
tissues(10, 11) . From their cDNAs, the amino acid
sequences for these proteins have been deduced (12, 13) . These binding proteins show extremely high
affinities (nanomolar) for folic acid, and somewhat lower affinities
for reduced folates and Mtx.
H]Mtx
(NHS-[
H]Mtx) to identify a 92-kDa glycoprotein
carrier (on a 7.5% gel) from Mtx transport up-regulated K562 cells
(designated K562.4CF). More recently, a highly glycosylated protein
(GP-Mtx), presumably representing the carrier, was isolated from
NHS[
H]Mtx-labeled membrane proteins from K562.4CF
cells on columns of Ricinus communis agglutinin
I-agarose(15) . A similar 80-85-kDa putative carrier was
identified in transport up-regulated CCRF-CEM cells (CEM-7A) by
affinity labeling with N
-(4amino-4-deoxy-10-methylpteroyl)-N
-(4-azido-5-[
I]iodosalicylyl)-L-lysine
(APA-[
I]ASA-Lys)(16) . Identical
labeling methods were used in murine L1210 cells to identify
presumptive transport proteins with molecular masses of 42-48 kDa
or 38 kDa (17, 18, 19) . Most recently,
homologous cDNAs from L1210 cells (20) and Chinese hamster
ovary (CHO) cells (21) were isolated and characterized. Both
cDNAs encoded 58-kDa proteins and, when expressed in transport-impaired
cells, restored Mtx sensitivities and Mtx transport activities. The
apparent discrepancies in the molecular masses of the cDNA-encoded
products and the membrane proteins identified in intact cells by
radioaffinity labeling have raised considerable interest in the
potential relationships among these proteins and their respective roles
in the overall scheme of carrier-mediated folate transport.
I]ASA-Lys, a known photoaffinity inhibitor
of the RFC. Taken together, the results strongly suggest that these
cDNAs encode the human RFC (hRFC).
Materials
[3`,5`,7-H]Mtx
(20 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA).
Unlabeled MTX and (6R,S)-5-formyltetrahydrofolate
(leucovorin) were provided from the Drug Development Branch, National
Cancer Institute, Bethesda, MD. Both labeled and unlabeled MTX were
purified prior to use as described previously(22) .
[
-
P]dCTP (3000 Ci/mmol) and
[
-
S-thio]dATP (1400 Ci/mmol) were obtained
from DuPont NEN. Fetal bovine serum and iron-supplemented calf serum
were from Life Technologies, Inc./BRL and Hyclone Laboratories, Inc.
(Logan, UT), respectively. The antifolate drug, 1843U89,
((S)-2-(5-(((1,2-dihydro-3-methyl-1-ocobenzo(F)quinazolin-9-yl)amino)1-oxo-2-isoindolinyl)glutaric
acid)) was provided by Burroughs Wellcome Company (Research Triangle
Park, NC). Sequencing reagents and enzymes were from United States
Biochemicals (Cleveland, OH). Restriction and modifying enzymes were
purchased from Life Technologies, Inc./BRL or Promega (Madison, WI).
Sequencing and PCR primers were obtained from Genosys Biotechnologies
Inc. (The Woodlands, TX).
Cell Culture
The K562 erythroleukemia line was
obtained from American Type Culture Collection (Rockville, MD). Wild
type K562, transport up-regulated K562.4CF, and transport
down-regulated K500E cells were cultured as described
previously(14) . Culture conditions for murine L1210 leukemia
cells were those of Schuetz et al.(18) .
3 and transport-defective Mtx-resistant (Mtx
RII Oua
2-4) cells(23) , gifts of Dr. Wayne
F. Flintoff (University of Western Ontario), were grown in
-minimal essential medium with 10% iron-supplemented bovine calf
serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). CHO
cells were grown as monolayers for transfection experiments and
cytotoxicity assays; for transport experiments, cells were grown in
suspension in spinner flasks. Transfected CHO clones were selected and
maintained in the presence of 1.5 mg/ml G418 (see below).
values were calculated
as the concentrations of inhibitors required to inhibit growth by 50%
compared to control cells grown under identical conditions, except that
growth inhibitors were omitted.
Isolation of Human cDNA Clones
A 5` fragment of
the murine putative RFC cDNA(20) , flanking positions 173 and
823 of the coding sequence, was prepared by reverse
transcription-polymerase chain reaction (RT-PCR) from mouse L1210 cell
total RNA. The oligonucleotides used were
5`-tccaggaattcgCCAAGGAGCAGGTGACTAACGAA-3` and
5`-cggtatcgatCCCACCAGAGGCACCAGAGAGACGC-3`. The EcoRI and ClaI restriction sites (lower case bases) were added to the up
and down-stream primers, respectively, for subcloning the PCR product
into the pBluescript SK(-) vector (Stratagene). RT-PCR was performed
using a Perkin-Elmer RT-PCR kit and an Ericomp thermal cycler. The
partial cDNA fragment, designated M-D6, was partially sequenced to
confirm its identity.
gt11 K562.4CF cDNA library was prepared
by Clontech (Palo Alto, CA). This library was plated at a density of
10,000 plaque-forming units/100-mm Petri dish and phage DNA was lifted
onto nylon filters (Magna Lift Nylon, MSI). The filters were
prehybridized in 50% formamide, 6
SSPE (1
SSPE
contained 0.15 M NaCl, 10 mM NaH
PO
, 1 mM EDTA, pH 7.4), 0.5%
SDS, 50 µg/ml denatured sheared salmon sperm DNA, and 5
Denhardt's solution for 2 h at 42 °C. Hybridization was
carried out at 42 °C overnight with the M-D6 probe radiolabeled
with [
-
P]dCTP by random priming. Filters
were washed (in 2
SSC (1
SSC contained 0.15 M NaCl, 15 mM Na
citrate, pH 7.0), 0.1% SDS for
20 min; twice at room temperature and twice at 42 °C), and positive
phage were identified by overnight autoradiography. From confirmed
positives, phage DNA was prepared using LambdaSorb phage adsorbent
(Promega) with the plate lysate method as described by the supplier.
Restriction mapping of phage DNA was performed, and cDNA inserts (KS6,
KS32, and KS43) were subcloned into the pBluescript SK(-) vector for
sequencing.
DNA Sequencing
Unidirectional nested deletion sets
were prepared from both sense and antisense KS32 cDNA/pBluescript
plasmid constructs using the Erase-a-Base system from Promega.
Single-stranded DNA templates were prepared from deletion set subclones
by infecting plasmid-containing XL-1 Blue cells with the helper phage
VCSM13 (Stratagene). Utilizing the universal T3 primer, both the sense
and antisense strands were sequenced completely using the
dideoxynucleotide chain termination method (24) with a
Sequenase Version 2 kit. Routinely, dGTP and dITP reactions were run in
parallel to avoid problems associated with compression caused by
G/C-rich sequences. Nucleotide sequence of the KS43 insert was obtained
primarily with primers designed from the KS32 sequence and
single-stranded template from the SalI-digested KS43 insert in
pBluescript. In addition, EcoRI fragments of KS43 and KS6 in
pBluescript were sequenced at both the 5`- and 3`-ends using T3 and T7
universal primers. Sequence data was analyzed using the University of
Wisconsin Genetic Computer Group (GCG) package. Homology searches in
nucleotide and protein sequence data bases were performed using the
BLAST network service at the National Center for Biotechnology
Information. Expression of KS32 and KS43 cDNA Inserts in CHO Mtx RII Oua 2-4 Cells-The KS32 and KS43 cDNA inserts in
pBluescript were directionally ligated into the expression vector
pcDNA3 (Invitrogen) utilizing the XbaI and KpnI
multi-cloning sites. The resulting plasmid constructs, designated pC32
and pC43, respectively, were used to transfect Mtx-resistant Mtx RII
Oua
2-4 cells; a mock transfection using the pcDNA3
vector was also performed. Transfection was carried out by the
polybrene procedure (25) using 2-4 µg of plasmid/3
10
cells in 60-mm Petri dishes. Seventy-two h post
transfection, cells were trypsinized, washed with Dulbecco's
phosphate-buffered saline (DPBS; 26), and replated at densities ranging
from 0.1 to 1
10
cells in 4 ml of fresh
-media
containing 1.5 mg/ml G418. G418-resistant CHO clones that exhibited
increased Mtx sensitivities over Mtx RII Oua
2-4
cells were selected for further analysis.
Membrane Transport Methodology
Logarithmically
growing cells were washed with DPBS and resuspended into Hank's
balanced salts solution. Transport experiments with
[H]Mtx were performed exactly as described
previously (14) using cell densities of 1-2
10
cells/ml. The levels of intracellular radioactivity were
expressed as picomoles/milligram protein or pmol/10
cells,
calculated from direct measurements of radioactivity and protein
contents of the cell homogenates. Protein assays were by the method of
Lowry et al.(27) . Intracellular accumulations were
corrected for surface adsorption as described by Sirotnak(2) .
Kinetic constants (i.e.K
, V
, and K
values)
for assorted folate and antifolate substrates were calculated from
Lineweaver-Burk and Dixon plots, respectively.
Northern Analysis
Total RNA was isolated from log
phase cells using the single step method of Chomczynski and
Nicoletta(28) . Total RNA from each cell line (20 µg) was
electrophoresed on 0.9% agarose gels containing 2.2 M formaldehyde and 1 MOPS buffer. The gel was equilibrated
in 10
SSC and capillary transferred to GeneScreen Plus membrane
(DuPont) in 10
SSC; the membrane was baked at 80 °C under
vacuum for 1.5 h. Membranes were prehybridized in QuickHyb solution
(Stratagene) for 15 min then hybridized for 1 h with the addition of
[
P]dCTP-labeled KS43 cDNA insert (labeled by
random priming). Nonspecific hybridization was removed by washing
membranes in 2
SSC, 0.1% SDS at 42 °C and, finally, in 0.1
SSC, O.1% SDS at 60 °C. After autoradiography, the filters
were stripped and rehybridized with a
[
P]dCTP-labeled
-actin cDNA probe
(Clontech). Densitometry of the autoradiograms was performed on a
Molecular Dynamics Computing Densitometer and analyzed using the
ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Cell Surface Labeling of the Transport Protein
A
photoaffinity analogue of Mtx, APA-[I]ASA-Lys,
was synthesized from the unlabeled compound under subdued light using
the procedure of Price and Freisheim(17) . The radiospecific
activity of the APA-[
I]ASA-Lys was 2.9
mCi/µmol. Cells (K562, wild type CHO, L1210, and the transfected
CHO lines) were incubated at a density of 1
10
cells/ml in ice-cold 20 mM HEPES, and 225 mM
sucrose (pH adjusted to 7.4 with MgO). Cells were irradiated with a
long wave-length UV lamp for 3 min, washed with DPBS (two times), and
the radiolabeled proteins extracted with 500 µl of 10 mM Tris-HCl, pH 7.0, 1% Triton X-100 in the presence of a proteolytic
inhibitor mixture(14) . Aliquots were electrophoresed on a 7.5%
denaturing gel(14) . The gel was dried and exposed to film.
Specificity of labeling was established by performing identical
incubations in the presence of 100 µM aminopterin (AMT).
Western Analysis with Anti-GP-Mtx Antiserum
Plasma
membranes from K562 wild type, CHO cells, and transfected clones were
prepared as described previously(15) . Membrane proteins
(50-100 µg) were electrophoresed on a 4-10% linear
gradient gel in the presence of SDS and electroblotted onto Immobilon P
(Millipore). Immunoblot analysis was performed as described previously
using rabbit antiserum to GP-Mtx, a putative glycoprotein component of
the hRFC(15) .
Isolation and Characterization of cDNA Clones
A
partial cDNA to the putative RFC from murine cells (M-D6) was
synthesized by RT-PCR and hybridized to a single 2.5-kilobase (kb)
transcript in L1210 total RNA(20) . Based on the increased
expression of a cross-hybridizing 3.1-kb transcript in K562.4CF over
wild type K562 cells in low stringency washes (2 SSC, 0.1% SDS
at 42 °C; not shown), a human K562.4CF cDNA library constructed in
gtll was screened with M-D6 under similar conditions.
Approximately 500,000 phage were screened, and eight clones were
selected from 40 positives. Phage DNAs were prepared from these eight
clones and restriction digest analysis revealed that all inserts
contained two identical SacI restriction fragments (300 and
800 base pairs (bp), gel not shown). The cDNA inserts for two clones,
KS6 and KS32 (1.4 and 2.5 kb, respectively), were subcloned into the EcoRI multicloning site of the pBluescript SK(-) vector. Four
other phage inserts contained an internal EcoRI restriction
site. The longest, KS43 (2.8 kb), was subcloned into pBluescript as two
separate EcoRI fragments (1.1 and 1.7 kb) and also in its
entirety as a SalI restriction fragment (a SalI
restriction site was present in the adaptors used in cDNA library
construction). The resulting plasmids were sequenced. The physical map
and sequencing strategies for clones KS6, KS32, and KS43 are shown in Fig. 1A.
The nucleotide sequence and the deduced
amino acid sequence for the KS43 cDNA insert is shown in Fig. 1B. The sequence contains a 98-bp 5`-untranslated
region, an open reading frame (ORF) of 1776 bp preceded by a Kozak
consensus sequence(29) , and a 3`-untranslated region of 864 bp
which is followed by a poly(A) sequence. The predicted molecular mass
of the translated protein is 65 kDa. One consensus N-glycosylation site was identified (at asparagine 58). For
all three clones (KS6, KS32, and KS43), the upstream 5`-noncoding
regions were highly heterogeneous starting at position -53 (Fig. 2). The nucleotide sequence of KS32 is identical to KS43
commencing with position -52, with the exception of a 626 base
pair deletion starting at position 1568 (underlined sequence in Fig. 1B) and an additional four bases (TGTG) in
the 3`-untranslated region immediately upstream from the poly(A) tail
at position 2718 (not shown in Fig. 1B). As a result of
the deletion in the 3`-ORF of KS32, the in-frame translational stop
codon (position 1774) is lost. The KS32 coding sequence continues into
the 3`-non-coding region with the utilization of a new stop codon at
position 2205 (Fig. 1B). The result is that the KS32
ORF contains 14 bp (including the stop codon; bold face in Fig. 1B) not used in KS43, encoding 4 new
carboxyl-terminal amino acids (LRCS). Clone KS6 appeared to be a
partial cDNA. Only the 5`- and 3`-ends of the KS6 insert were sequenced (Fig. 1A).
As expected, GenBank data base homology
searches identified the mouse (20; GenBank accession no. L23755) and
hamster (21; GenBank accession no. U03031) RFC sequences as highly
homologous to KS43. No other homologous sequence was identified at the
time this article was submitted. Amino acid sequence alignment analysis (Fig. 3), comparing the KS43 amino acid sequence with the
homologous mouse (20) and hamster (21) sequences,
revealed homologies of 79 and 80%, respectively; although the human RFC
contained 73-79 more amino acids. All 9 of the tryptophans
contained in the human sequence are conserved in the homologous hamster
and mouse RFCs. Likewise, 4 of 6 cysteines are conserved.
Interestingly, the single N-glycosylation site in the hRFC (at
asparagine 58) was only conserved in the hamster RFC, even though two
potential N-glycosylation sites were present in both the mouse
and hamster sequences(20, 21) . Computer analysis of
the deduced amino acid sequences, according to the criteria described
by Kyte and Doolittle (30; not shown), predicted multiple hydrophobic
domains similar to the homologous mouse and hamster
cDNAs(20, 21) .
KS43 was used as a probe for
Northern blots of total RNA from parental K562 cells, transport
up-regulated K562.4CF cells, and a variant line (K500E) with 70-fold
Mtx resistance associated with 82% impaired Mtx uptake(15) . A
major 3.1-kb transcript was detected in all three lines with an
intensity, in part, paralleling relative transport. Compared to wild
type K562 cells, the 3.1-kb transcript was 3.3-fold increased in
K562.4CF and 2.2-fold decreased in K500E cells (Fig. 4). A faint
4.2-kb transcript was also detected in the K562.4CF cells.
P]dCTP-labeled KS43 cDNA insert as described. Lane 1, wild type K562 cells; lane 2, Mtx transport
up-regulated K562.4CF cells; lane 3, Mtx transport impaired
K500E cells. Lower panel, the membrane was stripped and
reprobed with a [
P]dCTP-labeled mouse
-actin cDNA probe.
Expression of KS32 and KS43 cDNAs in Mtx Uptake-resistant
CHO Cells
To characterize the potential role of the human
cDNA-encoded RFCs in Mtx membrane transport, the KS43 and KS32
pBluescript plasmid constructs were digested with XbaI and KpnI and the inserts directionally ligated into the XbaI and KpnI multicloning sites of the expression
vector, pcDNA3. These pcDNA3 constructs, containing the KS43 and KS32
inserts and designated pC43 and pC32, respectively, were transfected
into Mtx-resistant Mtx RII Oua 2-4 CHO cells. Stable
transfectants were selected with G418 (1.5 mg/ml) and screened for Mtx
sensitivities over a range of 10-200 nM. Based on prior
reports of an enhanced membrane transport and increased cytotoxicity of
the benzoquinazoline antifolate, 1843U89, for human over murine
cells(3) , we assessed the growth inhibitory effects for this
compound, as well.
2-4 cells and
mock-transfected cells (designated pC29) were approximately 60-fold
resistant to Mtx compared to wild type CHO (Pro
3)
cells (Table 1). Mtx sensitivities for two transfectants, pC43/10
and pC32/2 (Table 1), are nearly identical to that of the
Pro
3 cells. Mtx RII Oua
2-4 cells
and mock transfected pC29 cells were completely insensitive to 1843U89,
up to 2000 nM. Sensitivities to 1843U89 for both pC43/10 and
pC32/2 transfectants were approximately 16-fold greater than for wild
type Pro
3 cells (Table 1) and only 2-fold less
than for the wild type K562 line (data not shown). These data strongly
suggest the presence of a functional human transport system in the
transfected CHO lines.
Initial rates of [H]Mtx
influx (at 0.5 µM) were assayed over 180 s in wild type
CHO, Mtx RII Oua
2-4, and the pC29, pC43/10 and
pC32/2 transfectants (Fig. 5). By this analysis, the initial
uptake rate for pC32/2 was not significantly different than wild type
CHO cells, whereas a 37% increase was observed for pC43/10. Neither Mtx
RII Oua
2-4 nor mock-transfected pC29 cells
accumulated significant [
H]Mtx over this
interval. Hence, both the full-length and 3`-truncated cDNAs
completely restored Mtx membrane transport.
H]Mtx by various CHO cell lines. Cells were
incubated at 37 °C, and 0.5 µM
[
H]Mtx uptake was measured as described under
``Experimental Procedures.''
Kinetic analyses of the
influx rates over a range of [H]Mtx
concentrations were performed; the kinetic constants were calculated
from Lineweaver-Burk plots and are summarized in Table 2. Whereas
wild type CHO cells exhibited a similar V
to
K562 cells, the K
for
[
H]Mtx binding was decreased greater than 6-fold (Table 2). Notably, the CHO lines transfected with either the
full-length (pC43/10) or 3`-truncated (pC32/2) human cDNAs had K
values more similar to the wild type
CHO than to K562 cells. The V
values for the
transfectants were similar to (pC32/2) or slightly higher (pC43/10)
than for the wild type lines. Due to their extremely poor Mtx uptake
(<3% of wild type), kinetic parameters for the Mtx RII Oua
2-4 CHO cells were impossible to measure accurately and,
therefore, are not included in Table 2.
To further
characterize the structural specificities of the restored transport
system in the CHO transfectants, experiments were performed to assess
the inhibitions of [H]Mtx influx by assorted
transport substrates (unlabeled Mtx, leucovorin, 1843U89, folic acid). K
values were calculated from Dixon plots
(not shown) and the K
and V
values in Table 2. K
values for these inhibitions are summarized in Table 3. A characteristic feature of the classical RFC from both
murine and human cells relates to increased affinities for Mtx and
reduced folates compared to folic acid (Table 3). Relative to
wild type CHO cells, [
H]Mtx transport in K562
cells exhibited a somewhat increased (
3-fold) sensitivity to
inhibition by 1843U89 and a markedly decreased (up to 19-fold)
inhibition by unlabeled Mtx. For Mtx, the calculated K
values approximated the K
values in Table 2.
The pC43/10
and pC32/2 transfectants exhibited transport characteristics of both the human and hamster cells. Hence, K values (Table 3) for Mtx were nearly identical to wild
type hamster cells. For 1843U89, the K
values are virtually identical with that for human K562 cells,
consistent with the results of the growth inhibition experiments (Table 1). For folic acid, the K
for pC43/10 transfectant was nearly identical to that for
wild type CHO cells; however, the K
for
the 3`-truncated clone, pC32/2, most resembled that for K562 cells. An
increased K
for leucovorin binding to the
pC32/2 transporter was measured, as well. The relative affinities of
the restored transport in the CHO transfectants for folic acid versus other transport substrates strongly suggest that the
uptake involves the classical RFC system. The differences in relative
substrate binding between the pC43/10 and pC32/2 transfectants
demonstrate that while the deletion of a terminal portion of the KS43
ORF results in some differences in the binding of particular transport
substrates, its effects on the overall transport properties of the
restored transport are minor. This is further considered below.
H]Mtx transport
in the pC43/10 and pC32/2 transfectants were accompanied by the
expression of hRFC transcripts (3.5 and 3.2 kb, respectively) on
Northern blots probed with KS43 insert (Fig. 6, lanes 4 and 5). When normalized to
-actin mRNA levels, the
hRFC mRNA level in pC43/10 was 35% higher than that of the pC32/2 line.
The KS43 cDNA probe did not hybridize to the homologous 2.5-kb hamster
transcript (21) in wild type CHO cells, nor was any signal
detected in either Mtx RII Oua
2-4 or
mock-transfected pC29 cells (Fig. 6, lanes 1-3,
respectively). There was a 13-18-fold disparity in the relative
expression of the hRFC transcript between the transfected CHO lines and
K562 cells (Fig. 6, lanes 4-6). Cell Surface Labeling of hRFC with
APA-[
I]ASA-Lys-Apparently identical
94-kDa surface proteins (Fig. 7) were specifically labeled in
wild type K562 cells and pC43/10 transfectants with
APA-[
I]ASA-Lys, a documented photoaffinity
inhibitor for the RFC(16, 17) . For the pC32/2
transfectant, an 85 kDa band was detected. The disparity between the
sizes of these
I-labeled bands in the CHO transfectants
and those predicted from the KS43/KS32 cDNA sequences (65 and 58 kDa)
were mostly, if not entirely, due to their N-glycosylation
(data not shown). Interestingly, the L1210 labeling control (Fig. 7) exhibited a band centered around 46 kDa, differing from
the predicted 58 kDa value from the published murine cDNA
sequence(20) ; this value is similar to that reported from
other laboratories by affinity
labeling(17, 18, 19) . No specific labeling
was detected in wild type CHO and the mock-transfected pC29 cells, even
after prolonged autoradiography.
P]dCTP-labeled KS43 cDNA insert as described. Lane 1, CHO wild type (Pro
3) cells; lane
2, Mtx RII Oua
2-4 cells; lane 3, clone
pC29; lane 4, clone pC43/10; lane 5, clone pC32/2;
and lane 6, wild type K562. Lower panel, membrane was
stripped and reprobed with a [
P]dCTP-labeled
mouse
-actin cDNA probe.
I]ASA-Lys. For each cell line, 1
10
cells were treated with 1 µM of
APA-[
I]ASA-Lys in the presence (+) or
absence(-) of 100 µM of AMT. Cells were irradiated,
washed with DPBS, and the radiolabeled proteins extracted with 500
µl of 10 µM Tris-HCl, pH 7.0, 1% Triton X-100. Equal
aliquots (based on starting cell numbers) were electrophoresed in the
presence of SDS. The figure shows an autoradiograph of the dried gel.
The molecular masses (in kDa) of standard proteins are
indicated.
In sharp contrast to the
13-18 fold differences in hRFC transcript levels between the
pC43/10 and pC32/2 CHO transfectants and wild type K562 cells (Fig. 6), affinity labeled hRFC protein levels were increased
only 1.7- and 3.4-fold higher than K562 cells, respectively (based on
densitometric readings normalized to actual micrograms of protein
loaded). This indicates that hRFC translation is relatively inefficient
in the transfected lines. The increased labeling of the pC32/2 cells
suggests that the truncated hRFC may be less efficient in mediating
transport (3-fold based on their respective V
values) than the form expressed in pC43/10 cells.
Do KS43 and KS32 cDNAs Encode for GP-Mtx?
Previous
studies from this laboratory described the characteristics and
isolation of a highly glycosylated membrane protein (i.e. GP-Mtx), following its radiolabeling with
NHS-[H]Mtx. Antiserum prepared to GP-Mtx in
rabbits detected a single membrane glycoprotein whose expression for a
series of K562 sublines paralleled Mtx transport over a 12-fold
range(15) . The possibility that hRFCs expressed in the pC43/10
and pC32/2 transfectants are identical to GP-MTX was directly
considered by Western blotting, using specific antiserum to GP-Mtx (15) . In this experiment, GP-Mtx was readily detected in
plasma membranes from wild type K562 cells; however, no immunoreactive
proteins were detected in either wild type CHO cells or the pC43/10 and
pC32/2 CHO transfectants (data not shown).
2-4 CHO cells. Mtx RII Oua
2-4
cells are characterized by severely defective Mtx transport and low
levels of surface [
H]Mtx binding(23) ,
associated with the expression of a non-functional hamster RFC
mRNA(21) . Moreover, CHO cells express no detectable membrane
folate-binding proteins(31, 32) . Restored transport
in the CHO transfectants appeared to involve the classical reduced
folate/Mtx transport system which has been extensively documented in a
variety of mammalian cells(4, 7) .
H]Mtx(14) . In the present report,
similar sized proteins were detected in the pC43/10 and pC32/2 CHO
transfectants, by photoaffinity labeling with
APA-[
I]ASA-Lys.
I]ASA-Lys.
H]Mtx uptake in the CHO transfectants exhibited both hamster and human characteristics. Based on K
values, up to a 4-fold increased
affinity for 1843U89 over wild type CHO cells (typical of human cells),
and a 19-fold increased affinity for Mtx over K562 cells (typical of
hamster cells) was observed. This anomaly may reflect differences in
protein folding, post-translational processing, and/or membrane
insertion of the hRFCs between hamster and human cells. Alternatively,
additional regulatory/modulatory factors may be required for the
expression of a human carrier system with a full spectrum of transport
substrate specificities. Similar concepts were previously advanced by
Underhill et al.(34) based on studies of reversion
analyses and somatic cell hybrids between transport-defective CHO lines
including the Mtx RII Oua
2-4 subline.
H]Mtx, and its expression on
Western blots approximated Mtx transport for a series of K562 sublines
over a 12-fold range(15) . GP-Mtx exhibited an apparent
molecular mass (92 kDa) on Western blots nearly identical to those of
the APA-[
I]ASA-Lys-labeled proteins in the CHO
transfectants. No immunoreactive proteins were detected with
anti-GP-Mtx in either pC43/10 or pC32/2 lines which expressed hRFC.
This strongly suggests that GP-Mtx and hRFC are two separate
glycoproteins with identical migration characteristics in SDS gels.
Alternatively, the same differences in post-translational processing
which may account for the hamster/human hybrid transport phenotype may
also render the hRFC isoforms expressed in hamster cells
non-immunoreactive with GP-Mtx antibodies.
(
)suggesting its peripheral
membrane localization(35) . A separate 38 kDa cytosolic or
peripheral membrane protein has also been implicated in RFC function in
CCRF-CEM and L1210 cells affinity labeled with
APA-[
I]ASA-Lys(16, 17) .
I]ASA-Lys, a documented
radioaffinity inhibitor of the RFC(16, 17) ,
confirming their capability to bind (and apparently transport)
antifolate substrates. Although the unusual kinetic profiles of the CHO
transfectants imply that additional factors may be required to manifest
the full spectrum of transport substrate specificities, these may also
be attributable to the host cell employed for our studies. We are
currently transfecting transport-impaired human cells with the hRFC
cDNAs to clarify this point. In any case, the availability of cDNAs
involved in the transport of Mtx and reduced folates in human cells
will undoubtedly facilitate future studies into the structure and
function of this critical membrane system and the molecular basis of
transport-mediated Mtx resistance.
I]ASA-Lys, N
-(4-amino-4-deoxy-10-methylpteroyl)-N
-(4-azido-5-[
I]iodosalicylyl)-L-lysine;
bp, base pair(s); CHO, Chinese hamster ovary; DPBS, Dulbecco's
phosphate-buffered-saline; hRFC, human reduced folate carrier; kb,
kilobase; NHS-[
H]Mtx, N-hydroxysuccinimide ester of
[
H]methotrexate; ORF, open reading frame; RFC,
reduced folate carrier; RT-PCR, reverse transcription-polymerase chain
reaction; MOPS, 4-morpholinepropanesulfonic acid.
3) and Mtx transport
defective Mtx RII Oua
2-4 CHO cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.