From the Department of Biochemistry and Molecular Biology, Medical
College of Ohio, Toledo, Ohio 43614-5804 and the
Department of Genetics, University of Pavia, Via
Abbiategrasso 207, 27100 Pavia, Italy
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ABSTRACT |
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The folate receptor (FR) type The cell surface receptor for folic acid (folate receptor,
FR)1 has the ability to
mediate physiologic folate uptake and to transport novel antifolate
drugs and folate conjugates (1-20). The receptor is a current major
focus as a tumor target for multiple experimental approaches in cancer
therapy. Several studies have shown that FR, when expressed at high
levels, could offer the preferred uptake route of novel classes of
antifolate drugs that target glycineamide ribonucleotide
formyltransferase and thymidylate synthase (3-6). Taking advantage of
the non-destructive nature of FR-mediated internalization of
folate-coupled macromolecules (7, 8), cytotoxins such as momordin,
pseudomonas exotoxin, and maytansinoids were shown to produce selective
killing in FR-rich cells (9-12). Furthermore, the toxicity of such
conjugates was dependent upon receptor density on the cell surface
(10). Folate-conjugated radiopharmaceuticals also appear to offer a
means of tumor imaging/radiation therapy (13-16). Folate-coated
liposomes were shown to selectively target FR-rich tumor cells (17) and
selective killing of the malignant cells was obtained by encapsulating
doxorubicin in the liposomes (18). By a similar strategy, it was
possible to deliver antisense oligonucleotides against the epidermal
growth factor receptor to FR-rich tumor cells (19). Furthermore,
selective folate-mediated targeting of an adenoviral vector to FR-rich
tumor cells has been achieved in the presence of an antibody to ablate the endogenous viral tropism (20).
The mechanism of folate uptake via FR has been shown to occur by an
endocytic process (7-8, 21-25). The three known isoforms of FR, types
FR isoforms are expressed in a tissue specific manner and are
selectively overexpressed in certain malignant tissues. FR- FR- may be
distinguished from FR-
by its higher affinity for the circulating
folate coenzyme, (6S)-5-methyltetrahydrofolate
(5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed
that a single leucine to alanine substitution at position 49 of the
mature protein sequence is responsible for the functional divergence of
FR-
(Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997)
Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-
to the
functional equivalent of FR-
should include amino acid substitution(s) downstream of residue 92 in addition to mutation of
L49A. To pinpoint those residues, chimeric
FR-
L49A/FR-
constructs including progressively
shorter segments of FR-
downstream of position 92 as well as
selected point mutants were studied. Simultaneous substitution of
Leu-49, Phe-104, and Gly-166 in FR-
with the corresponding FR-
residues Ala, Val, and Glu, respectively, reconstituted the ligand
binding characteristics of FR-
. The results also exclude a role for
other residues in FR-
in determining its functional divergence. A
homology model of FR-
based on the three-dimensional structure of
the chicken riboflavin-binding protein is used to show the position of
residues 49, 104, and 166 in relation to the hydrophobic cleft
corresponding to the riboflavin-binding pocket.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(26, 27),
(28), and
/
' (29), bind folic acid with a high
affinity (Kd < 1 nM) and a
stoichiometry of 1:1 (30). The mature proteins are 68-79% identical
in amino acid sequence, ranging in length from 220 to 236 amino acids. The proteins have either two (FR-
, FR-
) or three (FR-
) sites of N-glycosylation which cumulatively contribute to their
proper protein folding and intracellular trafficking but are not
required for folate binding (31). FR-
and FR-
are attached to the
cell surface by a glycosylphosphatidylinositol membrane anchor (26, 32-34), whereas, FR-
and a truncated form of the protein, FR-
' are constitutively secreted (35). The sites of
glycosylphosphatidylinositol modification in FRs-
and -
are
Ser209 and Asn211, respectively
(34).
is
specific for particular epithelial cells and is vastly up-regulated in
ovarian and uterine carcinomas (36-39). FR-
is not expressed at
significant levels in most normal tissues with the known exceptions of
placenta, spleen, thymus, and neutrophils (39, 40). Overexpression of
FR-
was observed in some malignancies of non-epithelial origin including myeloid leukemia (29, 40). FR-
and FR-
' are specific for hematopoietic tissues, particularly lymphoid cells (35).
and FR-
from both human (41) and murine (42) sources have
opposite stereospecificities for the reduced coenzyme forms of folate,
5-methyltetrahydrofolate (5-CH3H4folate) (Fig. 1) and 5-formyltetrahydrofolate
(5-CHOH4folate). In contrast to FR-
, FR-
preferentially binds to the unphysiologic
(6R)-diastereoisomers of these compounds relative to their
physiologic (6S)-diastereoisomers. FR-
also exhibits a
significantly lower affinity relative to FR-
for the
(6S)-diastereoisomer of
5-CH3H4folate, the major circulating form of
the vitamin. In addition, FR-
has a lower affinity for a variety of
antifolate compounds compared with FR-
(41).
View larger version (15K):
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Fig. 1.
Structures of the (6S)-
(top) and (6R)-
(bottom) diastereoisomers of
5-methyltetrahydrofolate
(N5-methyl-5,6,7,8-tetrahydropteroyl
glutamic acid).
The tissue specificity of FR isoforms and their elevation in malignant
tissues may be an important factor in selective targeting of malignant
cells via FR-mediated uptake of novel antifolates and folate
conjugates. It is therefore of significance to understand the
structural basis for the functional difference between the membrane
anchored FR isoforms. We have previously demonstrated a relatively
unambiguous approach to identify peptide segments or specific amino
acid residues that could account for the functional differences between
FR- and FR-
(43). The approach takes advantage of the fact that
FR-
and FR-
are structurally homologous, and therefore chimeric
constructs of the two proteins may be expected to result in functional
proteins that could be characterized; by systematic construction of a
series of such chimeras in which progressively shorter peptides of one
protein are substituted in the other protein, residues that are
significant for a specific functional difference may be mapped while
simultaneously excluding a role for other residues. In this manner we
have previously demonstrated that the functional divergence of FR-
from FR-
is due to Leu49 in FR-
(43); substitution of
Ala49 in FR-
with Leu conferred the ligand binding
characteristics of FR-
. However, conversion of FR-
to the
functional equivalent of FR-
could only be accomplished when, in
addition to the reciprocal mutation of L49A, the sequence downstream of
residue 92 in FR-
was substituted in FR-
. Thus, the functional
divergence of FR-
from FR-
could possibly be accounted for by one
or more residues downstream of position 92 in addition to
Ala49. The purpose of this study is to identify those
residues in FR-
.
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MATERIALS AND METHODS |
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Mutagenesis and Recombinant Plasmids--
Chimeric constructs of
FR- and FR-
were made by utilizing natural restriction sites or
by creating restriction sites by the polymerase chain reaction using
Vent DNA polymerase (New England Biolabs) and the cDNAs for FR-
or FR-
as template. Oligonucleotides (Life Technologies, Inc.) were
designed in two different ways in order to construct the appropriate
recombinant proteins. In cases where no suitable restriction site was
found, complementary primers containing an appropriate restriction site
or point mutation were used in conjunction with upstream and downstream
primers containing restriction sites. Alternatively, mutagenic
oligonucleotides were used as end primers to amplify the desired
fragment. All of the synthetic oligonucleotides (Table
I) were designed to contain restriction
sites or point mutations without undesirable alterations of amino acid
sequence. The polymerase chain reaction products were first digested at
both ends with the appropriate restriction enzymes and subcloned into
the plasmid pcDNA1 (Invitrogen) containing the
FR-
L49A mutant cDNA. The recombinant plasmid was amplified in Escherichia coli MC1061/p3 and purified using
the Qiagen plasmid kit (Qiagen). The entire cDNA sequence was
verified by dideoxy sequencing using AmpliCycle Sequencing Kit
(Perkin-Elmer).
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Cell Culture and Transfection--
Human 293 fibroblasts were
maintained in Eagle's minimal essential media (Irvine Scientific)
supplemented with fetal bovine serum (10% v/v), penicillin (100 units/ml), and streptomycin (100 µg/ml). The cells were transfected
using either LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's protocol or by the use of calcium phosphate (44).
Transfection efficiencies were normalized to a co-transfected
-galactosidase standard and found to be relatively uniform.
Preparation of Crude Plasma Membranes--
Crude plasma
membranes were prepared essentially as described (41). Confluent
cultures of transfected 293 cells were washed at 4 °C initially with
PBS (10 mM sodium phosphate, pH 7.5, 150 mM
NaCl) followed by acid wash (10 mM sodium acetate, pH 3.5, 150 mM NaCl) to remove endogenously bound folate from cell
surface receptors and a subsequent PBS wash. The cells were scraped off the plates and suspended in lysis buffer (1 mM
NaHCO3, 2 mM CaCl2, 5 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride, pH 7.7), incubated at 4 °C for 30 min and then frozen at
70 °C. The cells were then thawed and homogenized by 60 strokes in
a glass Dounce homogenizer. The homogenate was centrifuged at 1000 × g for 5 min to sediment nuclei and cell debris. The
supernatant was then centrifuged at 4000 × g for 45 min to sediment the membranes. The membranes were washed in cold
(4 °C) acid buffer by resuspending in the buffer using a 1-ml
syringe followed by sedimentation. After an additional wash in PBS the
membrane preparation was resuspended in PBS containing 1 mM
phenylmethylsulfonyl fluoride.
[3H]Folic Acid Binding Assay-- [3H]Folic acid (Morevek, specific radioactivity 37 Ci/mmol, 2.5 pmol/assay tube) was mixed with a membrane suspension in 0.5 ml of PBS and incubated for 60 min at room temperature on a rotary shaker. The membranes were sedimented at 12,000 × g for 30 min at 4 °C and washed once with PBS. The membranes were then dissolved in scintillation fluid (Eccoscint H, Fisher Scientific) and the associated radioactivity counted in a Beckman LS3801 liquid scintillation counter. Membranes from untransfected cells as well as membranes preincubated with excess unlabeled folic acid were used as negative control.
The [3H]folic acid binding assay for FR on whole cells in 6-well tissue culture dishes was performed as described previously (34). The cells were washed sequentially at 4 °C with PBS, acid buffer, and again with PBS. The cells were then incubated in a 1-ml solution of PBS containing 3 pmol of [3H]folic acid and 9 pmol of unlabeled folic acid and incubated for 30 min at 4 °C. The cells were then washed two times with PBS. Ice-cold acid buffer was used to remove bound folate and the amount of [3H]folic acid measured by liquid scintillation counting. Untransfected cells as well as membranes preincubated with excess unlabeled folic acid were used as negative controls.
Inhibition Studies-- The relative affinities of (6S)- or (6R)-diastereoisomers of 5-CH3H4folate for wild-type or mutant FRs were determined by measuring the IC50 values for the compounds for inhibition of [3H]folic acid binding using a range of reduced folate concentrations (1-500 nM). A fixed concentration (2 nM) of [3H]folic acid was used in these experiments. The assays were carried out as described above for the binding of [3H]folic acid to FR-rich membranes either in the absence of inhibitor or with simultaneous addition of inhibitor and [3H]folic acid. IC50 values were calculated using the Inplot computer program (GraphPad Software Inc., Version 4.03).
Structural Modeling--
A sequence alignment between cRBP and
FR- was performed using the program ClustalW (45). A homology model
of the conserved ligand-binding domain (peptide 12-188) was generated
with the modeling package Quanta (Molecular Simulations) using the cRBP crystal structure (46) as template. Nonconserved residue replacements were carried out using the mutated function within Quanta. The three
insertions all occur in the surface loop region, and their conformations were modeled using a main chain data base and side chain
rotamer search procedure in program O (47). The model was used for the
rotation and translation search in molecular replacement program Amore
(48) against the x-ray diffraction data of
FR-
.2 The resulting model
was further refined by rigid-body refinement and energy-minimization
calculations in X-PLOR (49). Finally, the stereochemical quality of the
model was confirmed using Procheck (50). The surface accessibility of
residues was calculated in Quanta using the Lee and Richards
algorithm (51) with the probe radius set as 1.4 Å.
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RESULTS AND DISCUSSION |
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To determine the role of individual amino acids of FR-
responsible for its functional variance from FR-
, the general
strategy adopted in this study was to examine the effect of
substituting peptides or amino acids of FR-
at corresponding
locations in FR-
. The basic premise of this approach is that one or
a combination of a few amino acids in FR-
are solely responsible for
its functional divergence and that they will similarly influence the
function of FR-
when substituted in it. For the results of such
studies to be most meaningful, it is also important that the mutations not interfere with protein folding and cell surface expression of a
functional receptor. Indeed it may be seen in the following sections
that all of the FR-
mutants were functional and showed expression
levels comparable to that of the wild-type protein. The focus of the
functional analysis of the mutant proteins was to test for increase in
affinity for (6S)-5-CH3H4folate and
absence of stereospecificity for
(6R)-5-CH3H4folate, both
distinguishing characteristics of FR-
. It was previously
demonstrated (43) that substitution of Ala49 in FR-
with
Leu, the corresponding residue in FR-
, conferred FR-
-like
characteristics. Conversion of FR-
to the functional equivalent of
FR-
by substituting the sequence downstream of position 92 of FR-
was only possible if Leu49 was simultaneously mutated to
Ala. Therefore in the following studies, all of the sequence
substitutions and mutations were made downstream of position 92 in
FR-
L49A.
Functional Mapping of FR- Peptides Downstream of Position
92--
cDNA constructs were made to encode chimeric proteins in
which portions of the carboxyl terminus downstream of amino acid 92 in
the FR-
L49A mutant were replaced by the corresponding
peptides from FR-
. The initial chimeric proteins generated were
FR-
1-139/
140-232(L49A) and
FR-
1-92/
93-139/
140-236(L49A)
(Fig. 2). The mutants were transiently
expressed in human 293 fibroblasts. Membrane preparations from the
transfected cells were tested for their relative affinities for the
(6S)- and (6R)-diastereoisomers of
5-CH3H4folate (Fig. 2). Both chimeric proteins
showed approximately 10-fold greater affinity for
(6S)-5-CH3H4folate than either the wild-type FR-
or FR-
L49A; however, the values were
approximately 4-fold lower than that of wild-type FR-
. These results
appeared to suggest that amino acid residues from both segments of
FR-
, i.e. peptides 93-139 and 140-232, would be
required to completely confer FR-
-like ligand binding
characteristics to FR-
L49A.
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Role of Divergent Amino Acids in Peptide 93-139 of FR---
To
further map the functionally relevant amino acid(s) within peptide
93-139, additional chimeric constructs in which shorter segments of
FR-
were incorporated into the FR-
L49A mutant were designed (Fig. 2) to produce:
FR-
1-92/
93-125/
126-236(L49A) and
FR-
1-92/
93-115/
116-236(L49A).
These two constructs showed functional characteristics similar to
FR-
1-92/
93-139/
140-236(L49A). Furthermore, chimeras in which peptides 126-232 and 116-232 of FR-
were substituted in FR-
L49A (Fig. 2) behaved similar to FR-
1-139/
140-232(L49A). It is therefore
reasonable to conclude that the peptide sequence 116-139 in FR-
has
little significance in the differential ligand specificities of the FR isoforms and that peptide 93-115 must contain functionally significant residues.
Among the 23 residues in peptide 93-115, only four, at positions 96, 104, 106, and 115, are unconserved in FR-. As noted previously, the
two known murine FR isoforms type 1 and type 2 correspond to the human
FR-
and FR-
, respectively, in terms of their ligand binding
characteristics. Alignment of the amino acid sequences of peptides
93-115 of FRs from both human and murine (52) sources (Fig.
3) indicates that amino acid residues at positions 96 and 106 are conserved in the murine FR isoforms. Furthermore, the amino acid at position 115 is the same (glutamic acid)
in both human FR-
and murine FR-1. Therefore if one assumes the
presence of analogous structure-function relationships among human and
murine FRs, it would appear that the likely candidates contributing to
functional differences between FR-
and FR-
do not occur at
positions 96, 106, and 115. Indeed, when these amino acids in
FR-
1-92/
93-115/
116-236(L49A)
were individually changed back to the corresponding residues of FR-
, it was apparent that the amino acids at positions 96, 106, and 115 were
inconsequential to the functional differences between FR-
and FR-
(Fig. 2); mutation of the amino acid at position 104, however, reverted
the characteristics of the chimera to those of FR-
L49A.
Furthermore, the double mutant, FR-
L49A,F104V, showed approximately 10-fold higher affinity for
(6S)-5-CH3H4folate compared with
FR-
(Fig. 2). These results clearly indicate that valine at position
104 in FR-
contributes partially to its distinct ligand binding
properties.
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Indentification of a Functionally Relevant Residue Downstream of
Position 139 in FR---
From the foregoing results it appears that
to achieve complete conversion of FR-
to the functional equivalent
of FR-
, it is necessary to substitute one or more residues
downstream of position 139 in addition to creating L49A and F104V
mutations in FR-
. To identify those residue(s), chimeras were first
constructed in which FR-
peptide sequences 140-155 or 156-232 were
substituted in the double mutant, FR-
L49A,F104V (Fig.
4).
FR-
1-139/
140-155/
156-236(L49A,F104V) showed properties similar to those of FR-
L49A,F104V
(Fig. 4). FR-
1-155/
156-232(L49A,F104V),
on the other hand, exhibited ligand binding specificity similar to
FR-
(Fig. 4) thus mapping the relevant amino acid(s) to the sequence
downstream of position 155. Since the carboxyl-terminal peptide
downstream of the glycosylphosphatidylinositol modification site
(Ser209) in FR-
is absent in the mature protein (34),
further mapping was restricted to peptide 156-208. Within this peptide
only seven residues are unconserved between FR-
and FR-
.
Alignment of the amino acid sequence of human and murine (52) FR
isoforms (Fig. 5) shows that only three
amino acids (positions 166, 167, and 191) are conserved between human
FR-
and murine FR-1 and between human FR-
and murine FR-2. It was
therefore undertaken to test the significance of those residues in
contributing to functional differences between FR-
and FR-
.
Mutation of Leu167 or Ser191 in
FR-
L49A,F104V to Ile and Pro, respectively, the
corresponding residues in FR-
, did not significantly alter the
properties of the protein (Fig. 4). However, when Gly166
was substituted with Glu, the corresponding residue in FR-
, the
ligand binding characteristics of FR-
were fully reconstituted in
FR-
L49A,F104V. Taken together, the results of this study
indicate that alanine at position 49, valine at position 104, and
glutamic acid at position 166 act synergistically to produce the
distinguishing ligand binding characteristics of FR-
compared with
FR-
.
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A Structural Model of FR--
FR shows considerable (~30%)
amino acid sequence identity with the 219-residue chicken
riboflavin-binding protein (cRBP) (53). The amino acid sequence
alignment of the two proteins has been reported (46). The conserved
amino acids include the 16 cysteine residues in FR and 6 tryptophan
residues. The nine pairs of cysteine residues in cRBP form disulfide
bridges to maintain its structural fold (54). In the three-dimensional
structure of cRBP, the conserved tryptophan residues at positions 61, 91, 117, 131, 135, and 168 and tyrosine 82 stack into a hydrophobic
pocket for binding the aromatic ring of the ligand (46). The sequence
similarity between FR and cRBP implies structural similarity between
these two protein families. We have built a homology model of the
folate-binding domain of FR- (Fig. 6)
based on the crystal structure of cRBP (46). In the model, residues 104 and 166 are 13.9 Å apart and are respectively located inside and at
the entrance of a cleft that corresponds to the ligand-binding pocket
in cRBP. Residue 49 is peripheral to this cleft, 24.3 Å away from
residue 104. The solvent accessible surface area of residues 49, 104, and 166 are 8.61, 27.5, and 91.7 Å2, respectively, and
their side chain fractional accessibilities are 0.11, 0.20, and 0.61, respectively. The model indicates that while conserved tryptophan and
tyrosine residues may contribute to the affinity of ligand binding,
residues 49, 104, and 166 may be expected to play either a direct or an
indirect role in causing the differential affinities and
stereospecificities of FR-
and FR-
for the coenzyme forms of
folate and for antifolate drugs.
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ACKNOWLEDGEMENTS |
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We thank Dr. Faming Zhang for help with modeling work and Michael Brun for assistance with graphics.
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FOOTNOTES |
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* This work was supported by National Institutes of Health, NCI, Grant R29CA57598 and a grant from Eli Lilly & Co. (to M. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed.
2 F. Zhang, M. Ratnam, J. Miller, C. Shih, and R. Schevitz, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FR, folate receptor; PBS, phosphate-buffered saline; H4folate, tetrahydrofolate; cRBP, chicken riboflavin-binding protein.
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REFERENCES |
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