From the Departments of Protein Chemistry,
§ Immunobiology, ¶ Biochemistry,
Analytical
Chemistry, ** Molecular Biology, and
Cell
Sciences, Immunex Corporation, Seattle, Washington 98101
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ABSTRACT |
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FLT3 ligand (FLT3L) stimulates primitive hematopoietic cells by binding to and activating the FLT3 receptor (FLT3R). We carried out a structure-activity study of human FLT3L in order to define the residues involved in receptor binding. We developed a rapid method to screen randomly mutagenized FLT3L using a FLT3R-Fc fusion protein to probe the relative binding activities of mutated ligand. Approximately 60,000 potential mutants were screened, and the DNA from 59 clones was sequenced. Thirty-one single amino acid substitutions at 24 positions of FLT3L either enhanced or reduced activity in receptor binding and cell proliferation assays. Eleven representative proteins were purified and analyzed for receptor affinity, specific activity, and physical properties. Receptor affinity and bioactivity were highly correlated. FLT3L affinity for receptor improved when four individual mutations that enhance FLT3L receptor affinity were combined in a single molecule. A model of FLT3L three-dimensional structure was generated based on sequence alignment and x-ray structure of macrophage colony-stimulating factor. Most residues implicated in receptor binding are widely dispersed in the primary structure of FLT3L, yet they localize to a surface patch in the tertiary model. A mutation that maps to and is predicted to disrupt the proposed dimerization interface between FLT3L monomers exhibits a Stokes radius that is concentration-dependent, suggesting that this mutation disrupts the FLT3L dimer.
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INTRODUCTION |
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FLT3 ligand (FLT3L)1 binds to and activates the cell surface tyrosine kinase receptor, FLT3R, initiating signaling events that regulate the proliferation and differentiation of multiple lineages of cells of the hematopoietic system (1-3). Alone, FLT3L stimulates little growth of highly purified bone marrow progenitor cells in vitro, yet when FLT3L is combined with other growth factors it has potent synergistic proliferative effects on these hematopoietic precursors (1, 4). The human FLT3R has been cloned and shown to belong to a subfamily of structurally related tyrosine kinase receptors that contain five extracellular Ig-like domains and an intracellular tyrosine kinase domain (5). Whereas FLT3R is expressed in a limited number of tissues, including human bone marrow, thymus, spleen, liver, and lymph nodes, FLT3L is widely expressed in human tissue (6, 7).
The human FLT3L gene encodes a 235-amino acid type I
transmembrane protein consisting of four domains: 1) an N-terminal
26-residue signal peptide, 2) a 156-residue extracellular domain, 3) a
23-amino acid transmembrane domain, and 4) a 30-residue cytoplasmic
domain (1, 2, 7). Soluble FLT3L is thought to be released into circulation from the cell membrane by protease cleavage (8). Soluble
FLT3L is a noncovalently linked dimer and contains six cysteine
residues that apparently form intramolecular disulfides. Karplus and
co-workers sorted the three-dimensional coordinates of nine-helical
bundle cytokines into three structural subfamilies: long, intermediate,
and short chain (9). Although the three-dimensional structure of human
FLT3L is unknown, sequence comparisons with other members of the
four-helix bundle protein family whose three-dimensional structures are
known, imply that it is a member of the left-handed short chain
antiparallel four-helix bundle cytokine structural subfamily (1). The
three-dimensional structure of five members of the short chain helical
cytokines have been solved, including interleukin-4,
granulocyte-macrophage colony-stimulating factor, interleukin-2, M-CSF,
and interleukin-5 (9). Structural features common to these proteins
include an up-up-down-down antiparallel topology for helices A-D and
two short strands of -sheet contained in the long cross-over
segments between helices A-B and C-D. The unique up-up-down-down
topology for helices A-D, similar genetic organization of exons and
introns, distant relatedness observed between some members, and similar
biological function indicate that helical bundle cytokines arose from
divergent evolution of a common ancestral protein. It is widely
believed that helical cytokines initiate signal transduction by
ligand-induced receptor oligomerization (10).
The current methods for site-directed mutagenesis are powerful tools for the study of structure and function of proteins and are highly efficient and effective when some idea of where to make the specific mutation is available. However, in the case of FLT3L, the protein region of interest is not well defined, so it is difficult to predict which mutation to make in order to bring about a desired change. The cross-reactivity of murine and human FLT3L for FLT3R (7) precludes the potential to identify residues of interest by swapping interspecies segments of polypeptide between these ligands. Comprehensive mutational studies of other four-helix bundle proteins may not be applicable to FLT3L because a number of these species are monomeric and bind class I hematopoietic receptors, whereas FLT3L is dimeric and binds to and activates a class III tyrosine kinase receptor. Therefore, the method employed in this study of FLT3L structure-function relationship is random mutagenesis of the DNA encoding FLT3L coupled with an activity-screening system.
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EXPERIMENTAL PROCEDURES |
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Plasmid Manipulation and DNA Sequencing--
The multicopy
expression vector paADH2 was described previously (11). This plasmid
has the 2 µ origin of replication, the ADH2 promoter to drive
expression of foreign genes, and the -factor leader to direct
secretion of heterologous proteins. Vector PIXY456 is derived from
paADH2 by the addition of a BamHI site adjacent to
the Asp718 site. Primers JM37
(AATTAGGTACCTTTGGATAAAAGACTCAGTGGGACCCAGGAC) and P11673
(ATATGGATCCCTACGGGGCTGTGGCCTCCAGGGGCCG) were used to amplify human
FLT3L from FLT3L-clone 9 cDNA (7). JM37 primer contains
an Asp718 site 20 bases from the 3'-end of the
-factor and fuses the
-factor leader to FLT3L. P11673 contains a
BamHI site for cloning into the PIXY456 expression vector.
The amplified fragment was digested with Asp718 and
BamHI and ligated to PIXY456 to construct PIXY771. The
Asn-Phe-Ser glycosylation site at positions 123-125 was mutated to
Asn-Phe-Ala using primer
JM40(CCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCGCCCGGTGCCTG). Primers JM40 and P11673 were used to amplify the 3'-end of the human FLT3L cDNA. This amplified fragment was digested with
BsaI and BamHI and used to replace the
BsaI-BamHI fragment in PIXY771, resulting in
plasmid PIXY797. Plasmid DNA was rescued from yeast clones (12) and was
sequenced with synthetic primers that would hybridize to the coding and
noncoding strands of the vector DNA 5' and 3' to the FLT3L
gene.
Mutagenesis and Transformation-- Mutagenic PCR was performed as described previously (14). Mutagenized FLT3L cDNA was introduced into the PIXY456 expression vector by homologous recombination (15). The PCR fragment has a 30-base pair segment 5' to and a 60-base pair segment 3' to the FLT3L gene that are homologous to the regions immediately adjacent to the gap of vector DNA. Transformants were selected on synthetic complete minus tryptophan medium (16).
Screening--
Transformed colonies, 0.5-2 mm in diameter, were
lifted onto cellulose acetate membranes. A second "capture"
membrane (nitrocellulose) was coated with 0.1 mg/ml monoclonal antibody
M5-FLT3L in 50 mM phosphate, pH 7.4, 150 mM
NaCl (PBS) for 16 h at 4 °C. The membrane was subsequently
blocked for 4 h at 4 °C in PBS containing 3% (w/v) bovine
serum albumin, 5% (w/v) nonfat dry milk (blocking solution) and then
washed twice in PBS. The wet capture nitrocellulose membrane was placed
on a YEPD plate (16), care taken to avoid air pockets, and covered in
turn with the cellulose acetate membrane with yeast colonies uppermost,
avoiding air pockets. The membrane sandwich was punctured with a needle
for later orientation, and the assembly was incubated at 30 °C for
18-22 h. The cellulose acetate membrane with colonies was removed and
placed on a fresh YEPD plate and stored at 4 °C for later recovery
of the yeast.
ELISA and Cell Proliferation Assay-- The ELISA used in this study was described previously (17). Murine WWF7 cells were assessed in [3H]thymidine incorporation assays, as described earlier (6).
BAF/B03 cells were engineered to express the full-length human FLT3R using the same procedure previously described (2) for producing BAF/B03 cells expressing the murine FLT3 receptor. Expression of human FLT3R by the cells was confirmed by examining the capacity of the cells to proliferate in response to soluble FLT3L and by flow cytometric analysis using biotinylated FLT3L.Recombinant Human FLT3L Preparation--
Wild type and mutant
FLT3L proteins were purified to greater than 90% homogeneity as
follows. 1.2 liters of yeast medium were filtered through a 0.22-µm
membrane, the pH was adjusted to 4.0 by the addition of glacial acetic
acid with rapid mixing, and the mixture was filtered through a
0.22-µm membrane again (conductivity 2-6 milliohms). This filtrate
was applied to a 30-ml Fractogel EMD SO3-650 (M) (EM
Separations) equilibrated with 25 mM NaCH3COO, 50 mM NaCl, pH 4.0, at 20 ml/min. Protein was eluted with
25 mM MES, 200 mM NaCl, pH 6.0. The pH was
adjusted to 7.5-8 by the addition of volume of 1 M Tris, pH 9.0, and the solution was then filtered through
a YM100 membrane (Amicon). FLT3L was affinity-purified by passing the
filtrate over a monoclonal antibody (M5
-FLT3L; Ref. 8;
CNBr-activated Sepharose 4B, Amersham Pharmacia Biotech) column
equilibrated in 50 mM NaHPO4, 300 mM NaCl, pH 7.4. Protein was eluted with 25 mM
NaHPO4, pH 11.3, and the eluant was neutralized by the
addition of
volume of 1 M monobasic phosphate.
The eluant was concentrated and applied to a Superdex 200 column
(Amersham Pharmacia Biotech) equilibrated in PBS at 2.5 ml/min. The
dimer peak was collected, concentrated, filtered through a 0.22-µm
membrane, and stored at 4 °C. The concentration of purified FLT3L
protein was determined by quantitative amino acid analysis after acid
hydrolysis. The level of contamination of purified protein was assessed
by running 2-4 µg of various FLT3L proteins on 16%
SDS-polyacrylamide gels and visualized with colloidal Coomassie stain
(Fig. 3). In all cases, the proteins were a single species (>90%)
with a Mr of 21,000.
Physical Characterization-- Gel filtration chromatography of purified proteins was performed on a 300 × 7.8-mm Bio-Sil 125-5 column (Bio-Rad) in PBS at a flow rate of 1 ml/min.
Circular dichroism spectra of proteins were obtained using a Jasco 500 C spectropolarimeter interfaced with an IBM AT computer. All spectra were measured in a 0.1-mm cell in PBS at 22 °C, between 260 and 195 nm using a 1-nm bandwidth and a 1-s time constant. The percentage of helical content was estimated as described previously by Sreerama and Woody (18).Radiolabeling, Binding Assays, and Data Analysis-- Purified recombinant FLT3L was labeled with 125I using a solid phase chloramine T analog (Pierce) to a specific radioactivity of 4 × 1014 cpm/mmol with no detectable loss of specific binding activity as assessed by inhibition assays with unlabeled FLT3L. Binding assays and data analysis were performed as described previously (19). Briefly, BAF-BO3 cells transfected with human FLT3R cDNA (0.5-1 × 106) were incubated with serial dilutions of 125I-FLT3L in binding media (RPMI 1640, 2.5% bovine serum albumin, 0.2% NaN3, 20 mM Hepes, pH 7.2) in 96-well microtiter plates maintained on a mini-orbital shaker (Bellco) at 37 °C for 90 min. Inhibition binding assays were carried out by holding the radiolabeled FLT3L concentration constant at 0.3 nM, while unlabeled competitor proteins were ranged from 150 to 0.001 nM.
Modeling of FLT3L-- We generated the quaternary FLT3L structure using FOLDER, a distance geometry-based method for homology modeling (20). FOLDER uses a sequence alignment between a template and model protein to identify residues in topologically equivalent positions. For topologically nonequivalent atoms such as variable loops and some side chains, chemical constraints, standard geometrical parameters, and chemical information like disulfide cross-links are used to compute a set of distances between these atoms, which is appended to the set of distances for topologically equivalent atoms. Distance geometry methods are a powerful technique for structure elucidation when modeling proteins with low sequence identity to the template. The x-ray crystallographic coordinates for M-CSF (21)2 served as the structural template. M-CSF is the most closely related protein to FLT3L, where a crystal structure has been solved. The amino acid sequence alignment between M-CSF and FLT3L described previously is based on genetic and structural constraints and is shown in Fig. 1 (1). FLT3L is a noncovalently linked dimer, and we modeled FLT3L with the assumption that the dimer interface would be the same in M-CSF and FLT3L. We used the computer graphics program InsightII to generate the images in Fig. 5.
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RESULTS |
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Isolation of FLT3L Mutants--
The screening protocol used in
this study is described in Fig. 2 and in
experimental procedures. We lifted yeast colonies transformed with
mutagenized FLT3L cDNA onto a cellulose acetate membrane, which was
then overlaid onto a nitrocellulose capture membrane previously coated
with a non-neutralizing antibody to FLT3L (M5FLT3L). Protein
secreted by yeast passed through the cellulose acetate membrane and was
captured by the M5
FLT3L immobilized on the nitrocellulose membrane.
The capture membrane was probed with human FLT3R-Fc followed by a
reporter reagent, horseradish peroxidase-goat anti-human IgG. The
receptor binding properties of the secreted FLT3L were assessed
visually by noting the variation in intensity of the stained spots
after the enzyme-linked reaction. Most colonies secreted FLT3L, which
stained similarly to protein secreted from yeast expressing wild type
FLT3L. About 1% of the colonies gave rise to spots devoid of stain
(Fig. 2A). We designated these clear white spots as receptor
binding deficient (FLT3L
) and isolated respective
individual yeast colonies by relating to their positions on the
cellulose acetate membrane. The appearance of stained spots whose
intensity was greater than that of wild type was a far rarer event
(Fig. 2B). We isolated these colonies and designated their
phenotypes FLT3L++, species that exhibit increased receptor
binding properties. In this manner, we screened approximately 60,000 colonies for receptor binding, with up to 300 colonies assayed per
standard size Petri dish (82-mm diameter). We isolated 214 FLT3L
colonies, representing only a portion of the
FLT3L
species, and 114 FLT3L++ species,
representing all of the detectable FLT3L++ colonies.
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Purification and Analysis of FLT3L Muteins: Receptor Binding and Cell Proliferation Activity-- We chose to purify all FLT3L muteins that exhibited greater than wild type specific activity because these are likely to be directly involved in receptor binding (H8Y, K84E, K84T, W118R, and Q122R). Proteins with specific activities lower than wild type were selected for further study due to their relative proximity to the mutations resulting in a FLT3L++ phenotype and their near wild type levels of expression (H8R, I11Y, F81S, K116E). We also selected L27P for purification for the reasons stated above.
The data in Fig. 4 clearly denote a strict correlation between relative biological activity and receptor binding affinity for this subset of purified FLT3L muteins. It is unknown if this strong correlation extends to other muteins listed in Table I. The Ka and the Kai for the wild type protein used in this study are 1.5 ± 0.7 × 1010 and 2.2 ± 0.7 × 1010 M
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Physical Characterization of FLT3L Muteins-- The results we present in Table III indicate an absence, with the exception of W118R, of gross structural perturbations for the subset of purified proteins. Monomeric unglycosylated FLT3L should have a molecular mass of 17,686 daltons. When analyzed by SDS-gel electrophoresis, yeast-produced FLT3L migrates at molecular mass of approximately 21 kDa due to the presence of core glycosylation at a single N-linked site (Fig. 4B). The Stokes radius as determined by size exclusion chromatography (Mr = 44,000) indicates that wild type and mutant proteins are dimeric. The helical content of wild type and mutant proteins as determined by circular dichroism are similar to that obtained for wild type protein. These results are expected, given that the near wild type levels of expression was an important criterion for choosing this subset of proteins. Previous studies have shown that misfolded proteins are degraded in the endoplasmic reticulum (22), suggesting that well expressed proteins secreted from yeast are in a stable fully folded form.
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DISCUSSION |
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We have generated a map of the FLT3L binding surface for its receptor using random mutagenesis and a functional screen. The data in Table III indicate that there is minimal gross structural perturbation, with the exception of W118R, in our collection of purified FLT3L muteins. These data suggest that most of the residues concerned may be directly involved in the energetics of receptor binding. Interpretation of the mutation pattern observed in this study (Fig. 3) is greatly facilitated by the three-dimensional homology model of FLT3L (Fig. 5).
The solved crystal structure of M-CSF was chosen as a template for modeling FLT3L, since several properties of M-CSF are most similar to FLT3L. M-CSF, and by analogy FLT3L and SCF, belong to the short chain helical bundle structural subfamily. Other members of this family share little identity, yet have remarkable structural similarity (9). FLT3L (1), SCF (23), and M-CSF (21) are dimeric, but unlike M-CSF, FLT3L, and SCF they are non-disulfide-linked dimers. The M-CSF dimerization interface was used to model the dimerization interface of FLT3L.
In addition to the sequence similarity between FLT3L and M-CSF, the FLT3L receptor-ligand system appears most similar to the M-CSF and SCF receptor-ligand systems. The receptors for FLT3L, SCF, and M-CSF are all type III tyrosine kinase receptors and share considerable sequence identity. The binding stoichiometry for polypeptide chains of SCF ligand with subunits of Kit receptor is 2:2 (24). Koths and colleagues (25) assert that M-CSF binds only one receptor chain per ligand polypeptide. These observations suggest that for SCF and M-CSF, and by analogy FLT3L, there is only one receptor binding site per ligand subunit and an overall 2:2 stoichiometry in the ligand-receptor complex.
The mutagenesis data and the three-dimensional model of FLT3L corroborate each other. The three hot spot regions (positions 8-15, 81-87, and 116-124), widely separated in the primary structure, cluster together in a small surface patch of the tertiary structure. The clustering of mutations in a small surface patch is consistent with a single receptor binding site per monomer as suggested by studies of similar receptor-ligand systems. In addition, the model indicates that some mutations map to the proposed dimer interface. Mutations that disrupt the dimerization interface would be predicted to shift the equilibrium from dimeric to monomeric FLT3L. The L27P mutation maps to this proposed dimerization interface and reduces the biological activity of the mutant protein. As predicted, the L27P mutant protein exhibits a concentration-dependent change in the apparent molecular weight consistent with an alteration in a monomer-dimer equilibrium (Fig. 6). In addition to L27P, we also identified another mutation that maps to the dimer interface, A64T. The A64T mutant protein was not purified or analyzed for its size exclusion properties. However, in the protein sequence alignment proposed by Hannum et al. (1), position Ala64 in FLT3L correlates with Phe67 in SCF. A mutation at Phe67 in SCF altered monomer-dimer equilibrium toward monomer and reduced the biological activity of SCF (23). The observation that the L27P mutation mapped to the dimerization interface and appeared to alter monomer-dimer equilibrium helped validate M-CSF as a template for modeling FLT3L structure.
As noted above, mutations that map to three hot spots scattered
throughout the primary sequence cluster together in a patch on the
FLT3L three-dimensional model (Fig. 5). His8 maps to the
center of this patch. We isolated two substitutions for histidine at
position 8, H8R, which is FLT3L, and H8Y, which is
FLT3L++. This histidine is conserved in murine FLT3L and
M-CSF (Fig. 1). Koths and co-workers (25) report that the activity of
M-CSF is reduced when the equivalent histidine, His9, is
substituted by alanine. High resolution three-dimensional analysis of
M-CSF mutant H9A/H15A shows no significant structural perturbations.
These data suggest that His9 of M-CSF and His8
of FLT3L are directly involved in the binding energetics with their
respective receptors. Lys84 is another basic residue of
particular interest. Removal of positively charged Lys84,
the penultimate residue of the C terminus of helix C, in muteins K84E
and K84T results in the FLT3L++ phenotype. The threonine
substitution of K84T is conservative with the serines found in the
equivalent site in M-CSF and murine FLT3L. This suggests that
Lys84 acts to diminish activity in native FLT3L by
destabilizing the interaction with FLT3 receptor.
Inspection of Fig. 5B reveals that K84E, Q122R, and H8Y form
a triangle of residues that enhance binding of FLT3L to its receptor. Residues that map within this triangle include Asp3,
Cys4, Ser5, and Gln7. We identified
the mutation D3G by subcloning a double mutant, but this mutation had
little influence on FLT3L biological activity (Table I). A previous
study showed that the C4S mutation reduced biological activity 10-fold
(26). Other residues that map within the triangle include
Ser5 and Gln7, which are Tyr5 and
Ser7 in murine FLT3L, respectively (Fig. 1). Since murine
FLT3L stimulates the human receptor, these residues are not critical
for receptor binding. These data suggest that residues that can be
changed to increase receptor affinity may include residues that may not be part of a continuous patch; another explanation is that this section
of the homology model is inaccurate. Consistent with the former
interpretation is the observation that W118R and L3H increase binding
affinity for the FLT3 receptor and introduce an extra positive charge
to the molecule. The W118R mutation disrupts some of the helical
content of the molecule (Table III), and Leu
3 is a
residue that is not normally found in the mammalian expressed molecule,
so it is unlikely that either residue is part of receptor binding site.
These results are consistent with a less direct effect of charge rather
than alterations in an area of the molecule critical for receptor
binding.
Double mutein K84E/Q122R maintains the close correlation between
receptor affinity and bioactivity that we observe for the individual
FLT3L++ mutations (Table II). This correlation breaks down
for quadruple FLT3L++ mutein,
L3H/H8Y/K84E/Q122R. Whereas receptor affinity for the quadruple mutant is over 8-fold greater than wild type, cell
proliferation activity is only 3-fold greater (Table II). Two recent
reports also indicate that the correlation between receptor affinity
and bioactivity diminishes as high affinity mutations are combined. Multiply substituted muteins of interleukin-6 (27) and ciliary neurotrophic factor (28) exhibit high affinity for their respective receptors but do not evince the same high levels of bioactivity. Two
explanations potentially account for the apparent limit to bioactivity.
First, Toniatti et al. (27) propose that receptor clustering
on the surface of cytokine-activated cells acts to increase local
receptor concentration to levels above the Kd of the
IL-6 muteins, thus limiting maximum bioactivity of the high affinity
mutants. Second, Reddy et al. (29) demonstrate that an
epidermal growth factor mutein with reduced receptor affinity exhibits
greater bioactivity than wild type ligand by diminishing two
attenuation mechanisms, endocytic internalization and degradation of
epidermal growth factor and receptor down-regulation. Therefore, the
maximum bioactivity of high affinity FLT3L muteins is potentially reduced by a rapid rate of ligand-mediated receptor endocytosis.
In summary, we have developed a novel method for screening a randomly mutagenized ligand in order to identify residues involved in receptor binding. This method should be applicable to ligand-receptor systems where appropriate receptor binding and biological assays are available. In addition, the ability to simply identify species with "improved" receptor affinity and bioactivity points toward a significant utility for this technology. The strength of this method is further enhanced when mutations can be correlated to a homology-modeled structure. The corroboration among primary, tertiary, and quaternary structural information provides a powerful indication that we have correctly mapped the functional epitopes of FLT3L.
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ACKNOWLEDGEMENTS |
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We thank Ginny Price, Randal Ketchum, and Doug Williams for critical reading of the manuscript and Sharon Reitman for excellent editorial comments. We are indebted to Sung-Hou Kim and Kirston Koths for providing the crystal coordinates for M-CSF. In addition, thanks go to Tammy Hollingsworth and Amy Weber for expert help in DNA sequencing and to Karen Longin for help with the WWF7 cell proliferation assays.
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FOOTNOTES |
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* 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: Dept. of Cell Sciences, Immunex Corp., 51 University St., Seattle, WA 98101. Tel.: 206-587-0430; Fax: 206-233-9733.
1 The abbreviations used are: FLT3L, FLT3 ligand; FLT3R, FLT3 receptor; hFLT3R, human FLT3 receptor; M-CSF, macrophage colony-stimulating factor; SCF, stem cell factor; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid; ELISA, enzyme-linked immunosorbent assay.
2 S.-H. Kim and K. Koths, personal communication.
3 S. D. Lyman, unpublished results.
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REFERENCES |
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