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Cloning, expression, and characterization of a novel guanylate-binding

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 Biochimica et Biophysica Acta 1384 Ž1998. 373–386

Cloning, expression, and characterization of a novel guanylate-binding

protein, GBP3 in murine erythroid progenitor cells
a,1
Byung Hee Han , Don Jae Park b, Robert W. Lim a , Jeong Hyok Im a , Hyun Dju Kim a,)

a
Department of Pharmacology, UniÕersity of Missouri-Columbia, School of Medicine, Columbia, MO 65212, USA
b
Department of Medicine, DiÕision of Hematology, Vanderbilt UniÕersity Medical Center, NashÕille, TN 37232-6305, USA
Received 14 October 1997; revised 20 February 1998; accepted 25 February 1998

Abstract

We report the molecular cloning of a novel guanylate-binding protein ŽGBP., termed mouse GBP3 ŽmGBP3. in Friend
virus-induced mouse erythroid progenitor ŽFVA. cells. The 71-kDa mGBP3 belongs to a family of known GBPs that
contain the first two consensus motifs, GXXXXGKŽSrT. and DXXG, but lack the third element, ŽNrT.KXD, found in
typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to
agarose-immobilized guanine nucleotides ŽGTP, GDP and GMP.. Moreover, mGBP3 has been found to have an intrinsic
GTPase activity with K m and Vmax values of 77 " 4 m M and 21 " 0.5 pmol miny1 m gy1 of protein, respectively. The
mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylationrmethylation motif CAAX at the carboxyl
terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3
transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The
mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to
an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of
guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this
paper has been submitted to the GenBanke with accession number U44731. q 1998 Elsevier Science B.V. All rights
reserved.

Keywords: cDNA sequence; GTPase activity; Gene expression; Subcellular localization; Erythropoietin; Interferon-g

Abbreviations: Epo, Erythropoietin; ERG, Erythropoietin-re- 1. Introduction

sponsive gene; FBS, Fetal bovine serum; FVA cells, Erythroid
progenitor cells isolated from the mouse spleen infected with
Friend virus; GBP, Guanylate-binding protein; His-mGBP3, histi-
dine-tagged mouse GBP3; IFN-gc, Interferon gamma; IMDM,
Iscoves, modified Dulbecco medium; RACE, Rapid amplification
of cDNA ends; RT-PCR, Reverse transcriptase-polymerase chain
reaction; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
)
Corresponding author. Fax: q1-573-884-4558; E-mail:
pharmhdk@muccmail.missouri.edu
1
Present address: Yuhan Research Center, Yuhan Pharmaceu-
tical Co., Seoul, South Korea.
GTP-binding proteins regulate a variety of cellular
activities including signal transduction, protein syn-
thesis, protein targeting, and cell motility Žreviewed
in Ref. w1x.. Amino acid sequence comparisons and
X-ray crystallographic data analysis of GTP-binding
proteins have revealed the presence of three hallmark
consensus elements, GXXXXGKŽSrT., DXXG, and
Ž N rT . K X D w 2 – 6 x . The first tw o m otifs
GXXXXGKŽSrT. and DXXG are known to interact

0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

PII S 0 1 6 7 - 4 8 3 8 Ž 9 8 . 0 0 0 3 4 - X
374 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386
with the phosphate groups of the guanine nucleotides,
whereas the third element ŽNrT.KXD is responsible
for the specificity of guanine recognition. For exam-
ple, the modification of Ž NrT.KXD motif by substi-
tuting Asp with Asn in p21ras w7,8x and EF-Tu w9,10x
leads to a loss of the guanine nucleotide binding
specificity.
A family of proteins termed the guanylate-binding
proteins ŽGBPs. have been originally described as
interferon ŽIFN. -inducible guanine nucleotide-bind-
ing proteins in fibroblasts and in macrophages w11,12x.
Molecular cloning of GBPs has uncovered several
characteristic features common to the GBP family
w13–16x. All GBPs possess the first two GTP-binding
consensus elements GXXXXGKŽSrT. and DXXG,
but lack the third ŽNrT.KXD motif. However, these
GBPs still retain a high degree of selectivity to
guanine nucleotides ŽGTP, GDP, and GMP. over
adenine and pyrimidine nucleotides w13,17x. Recently,
several GBPs have been shown to be GTPases with
slightly different catalytic properties. While the re-
combinant hGBP1 w17x hydrolyzes GTP predomi-
nantly to GMP, the chicken GBP w16x and the hGBP2
w18x also catalyze the conversion of GTP, but GDP is
the major product. In addition, most GBPs known to
date have an isoprenylationrmethylation modifica-
tion motif at their carboxyl termini. This posttrans-
lational modification promotes the ability of proteins
to associate with the plasma membranes w19–21x.
Both hGBP1 w17x and rat p67 GBP w15,22x have been
shown to be isoprenylated in vitro. The rat p67 GBP
has been reported to be localized in the plasma
membranes w15,22x. However, the physiological func-
tion of GBPs has not yet been elucidated.
Erythropoietin ŽEpo. is a hematopoietic growth
factor that regulates cell proliferation, differentiation
and apoptosis of erythroid progenitor cells w23–27x.
Erythroid progenitor ŽFVA. cells, isolated from the
spleens of mice infected with the anemia-inducing
strain of Friend virus, have been used as a cell model
system for the erythroid differentiationrmaturation
w28–34x. FVA cells undergo differentiation to
hemoglobin-rich reticulocytes in vitro within 48–72 h
upon exposure to Epo. It has been reported that Epo
induces early responsive genes such as MYC, MYB,
GATA1, BVL1, NFE2, and TAL1r SCL prior to the
expression of genes encoding erythrocyte-specific
proteins like globins w35–39x.
Recently, we reported the isolation of erythropoi-
etin-responsive genes ŽERGs. from the Epo-treated
FVA cells by differential hybridization w38x. In this
communication, we present our results in which we
cloned and characterized a novel 71-kD guanylate-bi-
nding protein, termed mouse GBP3 from the Epo-
stimulated FVA cell cDNA library. The mouse GBP3
protein shows a high sequence identity and GTPase
activity similar to those of the other known GBPs.
However, unlike other GBP3, the mGBP3 protein,
which lacks the isoprenylationrmethylation motif
CAAX, appears to be located in the cytosol of FVA
cells.
2. Materials and methods
2.1. Cloning and sequencing of mGBP3 cDNA
FVA cells were isolated from mouse spleen and
cultured as described w40x. The construction of the
murine cDNA library from Epo-stimulated FVA cells
and the isolation of ERGs have been described previ-
ously w38x. One ERG clone, erg11a, appeared to
encode part of a 2.6-kb transcript as determined by
Northern blot analysis Žsee below. . Using the w a y32
PxdCTP-labeled erg11a cDNA insert as a probe, a
random-primed lgt10 cDNA library, prepared from
polyŽ A.q RNA of FVA cells treated with 2 Urml
Epo for 1 h, was screened to isolate overlapping
clones as described w38x. Additional 5X sequence was
obtained by using the 5X-rapid amplification of cDNA
ends Ž RACE. protocol according to the method of
Frohman et al. w41x. Briefly, polyŽ A.q RNA isolated
from FVA cells were reverse-transcribed with an
erg11-gene specific reverse primer, 5X-AGGGCAT-
TCTCCAGGTACTC-3X corresponding to positions
654–673 of mGBP3 cDNA ŽFig. 1. . The first strand
cDNA was tailed with dATP by terminal deoxynu-
cleotidyl transferase ŽUSB. and amplified using an
erg11-gene specific primer, 5X-GACAAAGGTGCT
GCTCAGAAGCACAG-3X corresponding to positions
418–443, and a dT17-adapter primer with Taq DNA
polymerase ŽUSB. in a Amplitrone II thermal cycler
ŽBarnsteadrThermolyne. . The PCR product Ž; 0.5
kb. was gel-purified and subcloned into pGEM-T
vector ŽPromega. .
 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386 375

Fig. 1. The cDNA and deduced amino acid sequences of mouse GBP3. Ninety eight nucleotides at the 5X end of the cDNA were obtained
by the 5X-RACE PCR method. The rest of the sequence was derived from several overlapping clones containing the mouse GBP3 Ž erg11.
cDNA. Amino acid sequence was deduced from the longest open reading frame and indicated by the single letter code. Consensus motifs
for nucleotide binding are boxed. Two potential polyadenylation signals AATAAA, in the 3X-untranslated region, are marked by dotted
lines. Two repeats of the ATTTA sequence motif found in rapidly degraded messages are underlined. The bold-faced amino acids indicate
the residue used to generate polyclonal anti-mGBP3 antibody as described in Section 2.

All cDNA inserts were sequenced on both strands quences were analyzed and compared with those in
using a Sequenasee Version 2.0 DNA sequencing kit the GenBanke sequence data banks using a com-
ŽUSB. according to manufacturer’s protocol. Se- puter program provided by Genetics Computer Group.
376 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386
2.2. Northern analysis
Total RNA was isolated by the guanidium thio-
cyanate–phenol method w42x. RNA samples Ž 20
m grlane. were separated by electrophoresis in a
1.1% agarose gel containing formaldehyde, and trans-
ferred onto a nylon membrane Ž Amersham. . UV-
crosslinked membrane were prehybridized for 2 h at
658C in rapid hybridization buffer ŽAmersham. , and
hybridized in the same buffer overnight at 658C with
1 = 10 6 cpmrml of w32 Px-random-primed cDNA
probe. Blots were washed twice with 2 = SSC con-
taining 0.1% SDS for 20 min at room temperature
and twice with 0.2 = SSC containing 0.1% SDS for
20 min at 658C. Following the washes, blots were
exposed to X-ray film ŽKodak. at y708C with inten-
sifying screens.
2.3. RT-PCR
Two micrograms of total RNA were reverse-tran-
scribed for 1 h at 378C in a volume of 20 m l with
random primers. One tenth of the synthesized cDNA
was subjected to PCR amplification for the indicated
number of cycles. PCR conditions were as follows:
948C for 1 min, 568C for 1.5 min, and 728C for 1.5
min. The PCR products were separated by elec-
trophoresis in 1.5% agarose gels and visualized by
ethidium bromide staining. Under such conditions,
the RT-PCR procedure appeared to be quantitative,
since the yields of PCR products were proportional to
the amount of cDNA input Ždata not shown. . Primers
used for PCR were as follows: for mGBP3, 5X-TG-
GAGGCACCCATTTGTCTGGTG-3X and 5X-GAC
AAAGGTGCTGCTCAGAAGCACAG-3X ; for
mGBP1 gene, 5X-CAAGCTAGCTGGGAAGAG-
GACAGG-3X and 5X-GAACTTCCTGATACACAG-
GCGAGG-3X ; for mouse b-actin, 5X-TCCTATGTGG-
GTGACGAGGC-3X and 5X-CATGGCTGGGGTGT-
TGAAGG-3X.
2.4. Expression and purification of histidine-tagged
mGBP3
Histidine-tagged mGBP3 Ž His-mGBP3. was ex-
pressed using a baculovirus expression system. First,
the coding region of mouse GBP3 gene was gener-
ated by PCR using the following oligonucleotides:
5X-CGGAATTCCTCGAGGCACCCATTTGTCTG-
GTG-3X Žcorresponding to positions 89-112. and
5X-GCGGATCCTCTAGACTAACTACTTAGTGAG-
CC-3X Ž corresponding to positions 1923–1940. .
PCR-generated fragment was cloned into the pGEM
vector to form pGEM-mGBP3. The XhoIrSalI frag-
ment of the pGEM-mGBP3 was excised and ligated
into the corresponding site of pBlueBacHis2 Ž In-
vitrogen. . The resulting recombinant construct,
pBlueBacHis2-mGBP3 expresses a fusion peptide
containing mouse GBP3 amino acid residue 2–620
fused to the histidine tag at its N-terminus. Spodoptera
frugiperda Ž Sf9. cells were co-transfected with the
baculovirus Autographa california ŽAcMNPV. DNA
and the recombinant construct pBlueBacHis2-
mGBP3. The recombinant baculovirus was isolated
by plaque assay according to manufacture’s protocol
ŽInvitrogen.. Sf9 cells Ž2 = 10 6 cellsrml. were in-
fected with the recombinant baculovirus at a multi-
plicity of infection of 5 plaque-forming unit per cell
for 72 h.
His-mGBP3 was purified by nickel-chelation affin-
ity chromatography according to manufacturer’s pro-
tocol ŽQiagen.. Briefly, infected Sf9 cells were lysed
by sonication in binding buffer Ž20 mM Tris–HCl,
pH 7.9, 500 mM NaCl, 10% glycerol, 5 mM imida-
zole, 0.5% Triton X-100, 10 mM b-mercaptoethanol,
1 mM PMSF, 20 m grml of leupeptin and trypsin
inhibitor, and 5 m grml of pepstatin A.. Soluble
protein was loaded onto Ni–NTA resins ŽQiagen. and
washed with 15 bed volumes of the binding buffer.
His-mGBP3 was finally eluted with a step-wise gra-
dient of imidazole of 10 to 100 mM. Protein samples
were then applied to Q sepharose anion-exchange
chromatography column Ž Pharmacia. equilibrated
with a buffer containing 20 mM Tris–HCl, pH 8.0
and 0.1% Triton X-100. Proteins retained on the
column were eluted with a step-wise gradient of
NaCl of 10–300 mM.
2.5. Nucleotide binding assay
The purified His-mGBP3 fusion protein Ž; 5 m g.
was incubated with 30- m l bed volume of
nucleotide–agarose resins Ž Sigma. for 30 min at 48C
in 1-ml binding buffer as described previously w13x.
Resin-bound proteins were eluted with 20 m l SDS-
PAGE sample buffer containing 2% SDS by boiling
 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386 377

for 5 min. The eluted proteins were separated by tion fluid and the amount of radioactivity was deter-
SDS-PAGE in 10% polyacrylamide gels and stained mined using a scintillation counter ŽBeckman..
with Coomassie Blue.
2.7. Generation of polyclonal antibody and Western
2.6. GTPase assay blot

GTPase assay was performed as described w17x in a
buffer containing 20 mM Tris–HCl, pH 8.0, 100 mM
NaCl, 5 mM MgCl 2 , 1 mM DTT, 80 nM w a y32
PxGTP Ž 3000 Cirmmol. and unlabeled GTP Ž con-
centration as indicated in figures. at 378C. The reac-
tion was terminated with 1 mM EDTA and 0.5%
SDS. Samples were spotted on polyethyleneimine-
cellulose thin layer chromatography plates Ž Selecto
Scientific. and separated in solution containing 1.5 M
LiCl, 0.5 M acetic acid, and 0.025 M citric acid. The
dried plates were autoradiographed. Radioactive re-
gions on the TLC plates were scraped into scintilla-
Polyclonal antibody was raised by immunization
of rabbits with a synthetic peptide corresponding to
the mouse GBP3 amino acid residues 513–541. Of
the 29 residues present in this peptide, only 10–12
residues are conserved in other known GBPs Ž see
Fig. 2.. Rabbits were immunized with 100 m g of the
keyhole limpet hemocyanin-coupled peptide mixed
with complete Freund’s adjuvant, and boosted twice
with incomplete Friend’s adjuvant at a month inter-
val. For analysis of mouse GBP3 protein, protein
samples were separated by SDS-PAGE Ž 10% poly-
acrylamide gel. , transferred to nitrocellulose mem-

Fig. 2. Alignment of mouse GBP3 with other guanylate-binding proteins. The predicted amino acid sequence of mouse GBP3 gene was
aligned with that of mouse GBP1rmag1 w13,14x, mouse GBPmarmag2 ŽGenBanke accession number M81128., human GBP1 w13x, and
human GBP2 w13x. Gray area indicates that the corresponding amino acid is identical to that of mouse GBP3. Gaps Žy. are introduced to
maximize alignment. The boxed residues indicate the conserved GTP-binding consensus motifs, GXXXXGKŽSrT. and DXXG.
378 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386
branes, and preincubated with TBS Ž 20 mM Tris–
HCl, pH 7.5, 150 mM NaCl. containing 3% bovine
serum albumin. The blocked membranes were subse-
quently incubated with a 1:2000 dilution of poly-
clonal anti-mGBP3 antiserum, horseradish peroxi-
dase-conjugated goat anti-rabbit IgG, and visualized
with an enhanced chemiluminescence system Ž Pierce. .
2.8. Immunofluorescence staining
FVA cells were cultured in IMDM with 2 Urml
recombinant Epo. Twelve hours after the initiation of
the culture, the cells were rinsed twice with phos-
phate-buffered saline, fixed with 4% paraformal-
dehyde for 1 h at room temperature, and attached
onto coverslips. Fixed cells were permeabilized with
phosphate-buffered saline containing 0.1% Triton X-
100 for 15 min, and nonspecific reactive sites were
blocked with 10% goat serum. Cells were subjected
to sequential incubation with a 1:500 dilution of
polyclonal anti-mGBP3 antiserum, biotinylated goat
anti-mouse IgG antibody, followed by streptavidin-
conjugated Cy5e Ž Zymed Laboratory.. Immunofluo-
rescence image was analyzed by confocal fluores-
cence microscopy ŽBio-Rad model 600..
3. Results
3.1. Isolation of a mGBP3 cDNA clone
We screened a lgt10 cDNA library generated with
mRNA from Epo-treated FVA cells and isolated sev-
eral overlapping clones using the cDNA insert of
erg11a as a probe. Nucleotide sequences of the com-
posite erg11 cDNA were obtained from these over-
lapping clones with a total length of 2458 nu-
cleotides, in which 98 nucleotides at the 5X end were
generated by the 5X-RACE method.
Fig. 1 shows the nucleotide and deduced amino
acid sequences of the composite erg11 cDNA. The
amino acid sequence encoded by the longest open
reading frame of the cDNA predicts a protein of 620
amino acids with a calculated molecular mass of
70,720 Da. In the 3X-untranslated region, there are
two repeats of polyadenylation signal AATAAA and
two repeats of the ATTTA motif, which is commonly
found in rapidly degraded mRNA species.
Sequence comparison reveals that the putative pro-
tein encoded by erg11 clone bears resemblance to a
family of guanylate-binding proteins ŽGBPs. as shown
in Fig. 2. The overall sequence identity between the
protein encoded by the erg11 clone and the other
known GBPs varies from 52% to 62%. The protein
encoded by the erg11 clone is referred hereafter to as
mouse GBP3. The sequence conservation between
mouse GBP3 and the other GBPs is highest in the
region surrounding two nucleotide binding motifs, the
first GTP-binding motif GXXXXGKŽSrT., which is
also known as the Walker A motif, located at amino
acid residue 39–46, and the second GTP-binding
motif, DXXG at residue 91–94. Like the other GBPs,
the mouse GBP3 apparently lacks the third down-
stream motif, ŽNrT. KXD which is found in typical
GTP-binding proteins. However, unlike the other
GBPs, the predicted mouse GBP3 does not have a
CAAX motif at its C-terminus. Instead, the mouse
GBP3 has a hydrophobic 30-amino acid stretch at the
C-terminus not found in either GBP1 or GBP2. The
Fig. 3. Northern blot analysis of mouse GBP3 transcript. Twenty
micrograms of total RNA isolated from FVA cells were separated
by electrophoresis in a 1.1% agarose-formaldehyde gel, blotted
onto nitrocellulose membrane, and hybridized to w a y32 PxdCTP-
labeled probe containing mouse GBP3 cDNA residue 99-2458.
The hybridized membrane was exposed to X-ray films at y708C.
Lane 1 shows the ethidium bromide staining of rRNAs. Lane 2
represents the autoradiograph from the Northern analysis follow-
ing hybridization.
 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386 379

hydrophobicity profile of mGBP3, plotted by the
algorithm of Kyte and Doolittle w43x, is similar to that
of hGBP1 except for the hydrophobic region at the
C-terminus Ž data not shown..
3.2. Detection of mGBP3 mRNA by Northern analysis
contrast, mGBP3 transcript was not detectable by
Northern analysis in two erythroleukemia cell lines,
HCD-57 and SREI-2, and various mouse tissues in-
cluding brain, lung, heart, spleen, kidney, liver, and
intestine Ždata not shown. .

Northern analysis confirmed the expression of
mouse GBP3 gene in freshly harvested FVA cells
ŽFig. 3.. The mGBP3 cDNA probe corresponding to
nucleotide residues 99–2458 detected an approxi-
mately 2.6-kb transcript Žlane 2. in these cells. In
3.3. Determination of mouse GBP3 gene expression
by RT-PCR
Mouse GBP3 gene expression was examined fur-
ther by RT-PCR. Fig. 4A describes the changes in the

Fig. 4. RT-PCR analysis of mGBP3 expression in various mouse cells and tissues. Total RNA isolated from various sources were reverse
transcribed and amplified by PCR for various numbers of cycles indicated below. PCR products were separated by electrophoresis in a
1.5% agarose gel and visualized by ethidium bromide staining. RT-PCR of the constitutively expressed b-actin transcript was used as a
control for normalization of RNA input. ŽA. The GBP3 expression during FVA cell differentiation. RT-PCR was performed with total
RNA isolated from freshly isolated FVA cells Žlane 1. and cultured FVA cells in the presence of 2 Urml Epo for the indicated number of
hours Žlane 2–8.. The mGBP3 and b-actin transcripts were amplified for 27 cycles and 25 cycles, respectively. ŽB. The mGBP3 gene
expression in cell lines. mGBP3 gene expression was determined in FVA cells treated with 2 Urml Epo for 1 h Žlane 1., Epo-treated
erythroleukemia cell lines HCD-57 Žlane 2. and SREI-2 Žlane 3., thrombopoietin-treated megakaryocytes FDC-P2 Žlane 4., macrophages
RAW264.7 Žlane 5. and skeletal muscle cells C 2 C 12 Žlane 6.. mGBP3: 30 amplification cycles, b-actin: 25 amplification cycles. ŽC.
Tissue distribution of mGBP3 transcript. mGBP3 and b-actin transcripts were amplified for 30 cycles and 25 cycles, respectively. ŽD.
The mGBP3 gene induction by IFN-g . FVA cells Žlane 1–2. and macrophages Žlane 3–4. were treated with 10 Urml IFN-g for 1 h Žlane
2 and 4. or left untreated Žlane 1 and 3., and gene expression was determined by RT-PCR. For mGBP1 and mGBP3: 27 amplification
cycles in FVA cells Žlane 1 and 2., 35 cycles in macrophages Žlane 3 and 4.; for b-actin: 25 amplification cycles.
380 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386

mGBP3 mRNA expression levels in FVA cells un-
dergoing differentiation with Epo. It was found that
mGBP3 transcript expression was readily detectable
in freshly harvested FVA cells at the time of their
isolation from the spleen. In the course of erythroid
differentiation, the message levels steadily increased
to a maximum at 3 h, followed by a decrease to a
very low level by 24 h. By contrast, the expression
level of the constitutively expressed b-actin tran-
script remained unchanged for up to 24 h. In FVA
cells maintained without Epo, the mGBP3 transcript
expression levels also increased transitorily but to a
lesser extent compared to the message levels seen in
FVA cells with Epo Ž data not shown..
Fig. 4B shows the expression levels of mGBP3
transcript determined in various cell lines. It is evi-
dent that the Epo-treated FVA cells displayed by far
the highest level of mGBP3 transcript expression.
The mGBP3 transcript was also detectable in the
thrombopoietin-stimulated megakaryocytes, FDC-P2.
On the other hand, the mGBP3 mRNA was either
expressed at low levels or not detectable in other cell
lines, including the erythroleukemia cell lines HCD-
57 and SREI-2, the macrophages RAW264.7, and the
skeletal muscle cells C 2 C 12 . In addition, the mGBP3
transcript was expressed to a varying degree in nu-
merous mouse tissues including the lung, spleen,
heart and kidney ŽFig. 4C. .
Since IFN-g has been known to induce other GBP
transcripts, it was of interest to determine whether
IFN-g can also induce mGBP3 gene expression. Fig.
4D shows that, in addition to mGBP3 transcript, FVA
cells also expressed mGBP1 transcript to a high basal
level. The treatment of FVA cells with IFN-g re-
sulted in a small induction of both transcripts. In
contrast, the basal levels of expression of both GBP
transcripts were low in the macrophages RAW264.7,
and required increased number of PCR amplification
cycles for their detection Ž Fig. 4D, lane 3.. Upon
treatment with IFN-g , both GBP transcripts were
highly induced Ž Fig. 4D, lane 4. .
3.4. Expression and purification of His-mGBP3
To investigate biochemical properties of mouse
GBP3, we isolated recombinant mGBP3 protein us-
ing a baculovirus expression system. Infection of Sf9
cells with the recombinant baculovirus carrying the
His-mGBP3 insert resulted in an expression of histi-
dine-tagged mGBP3 protein. The fusion protein con-
sisted of mGBP3 amino acid residue 2–620 and
additional 38 amino acids at the N-terminus, includ-

Fig. 5. Expression and purification of histidine-tagged mouse GBP3 in Sf9 cells. ŽA. Sf9 cells were infected with recombinant
baculovirus carrying the histidine-tagged mGBP3 for 72 h. Aliquots from various stages of purification by Ni-chelation affinity column
were analyzed by SDS-PAGE and Coomassie Blue staining. Lane 1, soluble protein fraction from non-infected Sf9 cells; lane 2, soluble
protein fraction from infected Sf9 cells; lane 3, Ni-chelation column flow through; lane 4, fraction washed with binding buffer; lane 5,
fraction eluted with 5 mM imidazole; lane 6, fraction eluted with 30 mM imidazole; lane 7, eluted with 100 mM imidazole; lane 8,
molecular mass markers. ŽB. SDS-PAGE analysis of His-mGBP3 Ž; 5 m g. purified by Q sepharose anion-exchange chromatography
Žlane 1..
 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386 381

ing the histidine tag which is necessary for the pro-

tein purification by Ni-chelation affinity chromatog-
raphy. Optimal His-mGBP3 expression was observed
when Sf9 cells were infected with the recombinant
baculovirus at the multiplicity of infection of 5
plaque-forming unit per cell for 72 h.
Fig. 5A displays SDS-PAGE of the proteins ob-
tained from various steps during the fusion protein
purification. Total soluble protein fractions prepared
from non-infected and infected SF9 cells are shown
in lane 1 and lane 2, respectively. The bulk of the
soluble proteins from the infected Sf9 cells were not
retained by the Ni-chelation affinity column Ž lane 3. .
Increasing the imidazole concentration in the elution
buffer differentially eluted proteins of varying sizes
Žlane 4–6.. A highly pure His-mGBP3 protein was
obtained by elution with 100 mM imidazole Ž lane 7..
The yield of His-mGBP3 was approximately 5–10
mgrl of Sf9 cell culture. To obtain His-mGBP3 of
higher purity, the affinity-purified protein was subse-
quently further purified by anion-exchange chro-
matography. His-mGBP3 was retained on the anion-
exchange column after loading and washing, and was
eluted with buffer containing 300 mM NaCl Ž Fig. 5B,
lane 1.. The purified protein showed an estimated
molecular weight of 75 kDa, and was immmunologi-
cally reactive to the polyclonal anti-mGBP3 antibody
Ždata not shown. .
3.5. Nucleotide binding actiÕity of His-mGBP3
Fig. 6 shows the binding activity of His-mGBP3 to
various nucleotide-coupled agarose resins. Equal
Fig. 7. Determination of kinetics for GTPase activity of His-
mGBP3. His-mGBP3 Ž0.2 m gr m l. was incubated at 378C for 20
min with various concentrations of GTP. GTP hydrolysis was
analyzed by polyethyleneimine cellulose thin layer chromatogra-
phy and autoradiography. The double-reciprocal plot of the sub-
strate vs. GTP hydrolysis rate is shown. Data represent the
mean"S.D. from three independent experiments. The calculated
K m and Vmax values are 77"4 m M and 21"0.5 pmol miny1
m gy1 of protein, respectively.
aliquots of the affinity purified His-mGBP3 were
incubated with nucleotide–agarose resins, and the
retained proteins were eluted with the SDS-contain-
ing buffer and analyzed by SDS-PAGE. Fig. 6 lane 2
shows Coomassie Blue staining of the aliquot of
purified mGBP3 used as control in the absence of
nucleotide–agarose resin. It is evident that a sizable
fraction of the initial starting mGBP3 was bound to
agarose-immobilized guanine nucleotides GTP, GDP,
or GMP. By contrast, protein retention to agarose-im-
mobilized adenine nucleotides ŽATP, ADP, and AMP.
and pyrimidine nucleotides ŽCDP, CMP, UDP, and
UMP. was negligible. The His-mGBP3 also did not
bind to the protein A-agarose resin.

Fig. 6. Nucleotide binding assay of His-tagged mGBP3. Purified His-mGBP3 protein was incubated with the nucleotide-agarose resins
indicated for 30 min at 48C. The resins were washed with the binding buffer, and the bound proteins were eluted with PAGE sample
buffer containing 2% SDS. Samples were analyzed by SDS-PAGE and Coomassie Blue staining. Lane 1, molecular mass markers; Lane
2, the total amount of His-mGBP3 protein used as starting material in the binding reaction; Lane 3, protein retained by protein-A agarose
resin, lane 4–13, protein bound to indicated nucleotide–agarose resins.
382 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386

3.6. GTPase actiÕity of His-mGBP3

Fig. 8. GTP hydrolysis by His-mGBP3 in the presence of various
nucleotides. His-mGBP3 Ž0.18 m gr m l. was incubated at 378C in
a buffer containing 80 nM w a y32 PxGTP, 50 m M GTP, and 2
mM competitor nucleotides indicated. Hydrolysis of the radiola-
beled GTP was analyzed by polyethyleneimine cellulose thin
layer chromatography and autoradiography. Data represent the
mean"S.D. from three independent experiments.
We found that the purified His-mGBP3 has GT-
Pase activity that catalyzes conversion of GTP to
GDP Ždata not shown. . Maximal GTP hydrolysis
activity was correlated with the peak of His-mGBP3
elution during anion-exchange chromatography,
showing that GTPase activity measured is likely to
have stemmed from His-mGBP3. The GTP hydroly-
sis was dependent on magnesium and the optimum
pH of the reaction was 8.0. To determine K m and
Vmax , the GTPase reaction was carried out in the
presence of varying GTP from 15 to 180 m M. At
these substrate concentrations, GTP hydrolysis was
linear up to 40 min Ždata not shown. . Fig. 7 displays
double-reciprocal plot of GTP concentration vs. GTP
hydrolysis rate. The K m and Vmax values for the

Fig. 9. Immunofluorescence localization of mouse GBP3 protein in FVA cells. Panels A and C show bright-field images of FVA cells
cultured for 12 h with erythropoietin. Panel B and D show confocal immunofluorescence images of the same fields corresponding to A
and C, respectively. Cells were treated with pre-immune serum Žpanels A and B. or polyclonal anti-mouse GBP3 antiserum Žpanels C and
D., followed by incubation with biotinylated goat anti-mouse IgG and Cy5-conjugated streptavidin ŽOriginal image= 600..
 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386
GTPase activity of His-mGBP3 were estimated to be
77 " 4 m M and 21 " 0.5 pmol miny1 m gy1 of pro-
tein, respectively, from three experiments using a
single His-mGBP3 preparation. The specific activities
of GTPase, however, varied in different preparations.
To determine the substrate specificity of His-
mGBP3, GTPase assay was performed with His-
mGBP3 in the presence of 80 nM w a y32 PxGTP, 50
m M unlabeled GTP, and 40-fold molar excess of
competitor nucleotides Ž Fig. 8. . Hydrolysis of the
radiolabeled GTP was inhibited by guanine nu-
cleotides in the decreasing order of GTP, GMP, and
GDP. By contrast, ATP and CTP had little effect on
GTP hydrolysis by His-mGBP3.
3.7. Subcellular distribution of mGBP3
Fig. 9 displays immunofluorescence localization of
mGBP3 in FVA cells cultured for 12 h with Epo.
Consistent with previous observation w33x, the FVA
cells appear to consist of a large prominent nucleus
surrounded by a small region of cytoplasm between
the nucleus and the plasma membrane Ž Fig. 9A,C..
The immunofluorescence staining by anti-mGBP3 an-
tiserum showed a preferential distribution of mGBP3
to this non-nuclear compartmental edge, presumably
the cytosol ŽFig. 9D. . Moreover, the immunostaining
appears to be more prominent in the larger, less
matured erythroid progenitor cells than in the smaller,
more differentiated cells. No specific immunofluo-
rescence could be detected in FVA cells when the
primary antiserum was substituted with pre-immune
serum Ž Fig. 9B. . The localization of mGBP3 protein
was also examined by SDS-PAGE and immunoblot
assay on subcellular fractions. Anti-mGBP3 anti-
serum recognized exclusively a protein of approxi-
mately 70 kDa in all cellular fractions including
membrane, cytosol and nucleus Ž data not shown. .
The absence of cross-reaction to proteins of other
molecular sizes suggests that the antiserum is specific
to GBP3 and does not recognize other known GBPs.
In addition, the immunoblot data confirm that at least
a majority of mGMP3 is present in the cytosol of the
FVA cells. However, since we have not determined
to what extent the membrane and nuclear fractions
might be contaminated by cytosol, the possible pres-
ence of mGBP3 in membrane and nuclear compart-
ments should be viewed with caution.
383
4. Discussion
GBPs were originally identified as a group of
proteins with a strong binding activity to GMP-
agarose resins w11,12x. The stimulation of human
fibroblasts and murine macrophages by interferons
resulted in the synthesis of GBP1 along with other
minor GBPs. The molecular cloning and sequencing
of two members of genes encoding GBPs have re-
vealed salient features common to GBP family pro-
teins w13–16x. All members of this GBP family have
the first two of three consensus elements found in
typical GTP-binding proteins. Despite the lack of the
third consensus GTP-binding motif Ž NrT. KXD,
which is believed to be essential for the guanine
recognition, GBPs exhibits the nucleotide binding
specificity to guanine nucleotides Ž GTP, GDP, and
GMP. . In addition, GBPs exhibit unusual GTPase
activities that hydrolyze GTP to GMP or GDP w15–
18x. Although GBPs are now a rapidly growing fam-
ily of GTP-binding proteins, their physiologic role
has yet to be elucidated.
In the present study, we have cloned the cDNA
encoding a novel GBP termed mouse GBP3 in FVA
cells. Like the other known GBPs, the mouse GBP3
possesses only the first two GTP-binding consensus
motifs GXXXXGKŽSrT. and DXXG but devoid of
the third consensus element Ž NrT.KXD. The hy-
drophobicity profile of mouse GBP3 is similar to that
of hGBP1 except for the C-terminus, which has an
hydrophobic stretch of 30 amino acid residue. Taken
together, these findings suggest that mouse GBP3
with a predicted molecular mass of 71 kDa is a new
member of the GBP family. In response to stimula-
tion by IFNs, mouse embryonic and splenic cells are
known to express multiple GBPs with molecular
masses of 65 kDa Ž which was identified as mGBP1. ,
70 kDa, and 71 kDa w12x. Inasmuch as amino acid
sequence information for the 70- and 71-kDa GBP is
currently unavailable, it is not known whether the
mouse GBP3 we cloned is identical to the 70- and
71-kDa proteins. Moreover, based on the partial se-
quence of human GBP3 currently available w44x,
mGBP3 is unlikely to be the murine homolog of the
human GBP3.
To characterize the biochemical property of mouse
GBP3, we initially expressed His-GBP3 fusion pro-
tein using a bacterial expression system. Although the
384 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386
recombinant His-mGBP3 protein could be expressed,
it was unstable undergoing extensive degradation dur-
ing its expression and purification Ždata not shown..
In contrast, the expression of the His-mGBP3 protein
in the Sf9 insect cells using a baculovirus expression
system allowed the isolation of the highly purified
fusion protein by nickel-chelation affinity chromatog-
raphy and by anion-exchange chromatography. His-
mGBP3 is similar to the other GBPs in that His-
mGBP3 strongly binds only to guanine nucleotides
ŽGTP, GDP, and GMP.. Schwemmle et al. w16x postu-
lated that the TVRD sequence, which is conserved in
other known GBPs, might be responsible for retain-
ing the guanine nucleotide-binding specificity. In
mouse GBP3, the sequence corresponding to TVRD
is substituted by AVRD at amino acid residue of
173–176. Thus, the substitution of Thr 173 to Ala173
does not seem to affect the nucleotide binding speci-
ficity of mouse GBP3.
The mouse GBP3 differs from the other GBPs in
that the C-terminal CAAX motif, known for the
post-translational modification signal via isoprenyla-
tionrmethylation, is absent. It is now known that the
isoprenylationrmethylation modification plays a cru-
cial role in enhancing the association of the p21ras
family proteins and the g-subunits of the G-proteins
with the plasma membranes w19–21x. Both hGBP1
w17x and rat p67 GBP w15,22x have been shown to be
isoprenylated in vitro at the carboxyl terminal Cys
residue. Isoprenylated p67 GBP appeared to be pre-
dominantly associated with the cell membranes in rat
smooth muscle cells w15x. By contrast, despite being
isoprenylated, human GBP1 has been recently re-
ported to be localized in the cytosol of the promyelo-
cytic cell line HL-60 w45x. We also found the pres-
ence of mGBP3 in the cytosol of FVA cells based on
immunofluorescence staining using a polyclonal
anti-mGBP3 antibody Ž Fig. 9. and by the subcellular
fractionation and immunoblot analysis Ž unpublished
observation. .
Since the cDNA encoding mouse GBP3 was ini-
tially identified in Epo-stimulated FVA cells, we
examined the expression of mouse GBP3 transcript in
the course of erythroid differentiation. FVA cells
represent the colony-forming unit-erythroid Ž CFU-E.
stage of erythroid development, requiring Epo for
proliferation and differentiation into enucleated
reticulocytes or erythrocytes. We found that the mouse
GBP3 transcript is already expressed at a high level
in FVA cells at the time of their isolation from the
spleen. In point of fact, mGBP3 expression level is
higher in FVA cells than in other cells we tested thus
far ŽFig. 4.. Although mouse GBP3 mRNA is tran-
siently increased within 3 h, and then down-regulated
thereafter in FVA cells undergoing differentiation in
the presence of Epo, the induction of the mouse
GBP3 transcript is not strictly Epo-dependent. A
transient increase in mouse GBP3 transcript is also
observed in FVA cells cultured in Epo-deprived cul-
ture medium, but to a lesser extent than in Epo-ex-
posed cells Ždata not shown.. The reasons for the
high ‘basal’ expression of mGBP3 in FVA cells is
not yet known. It is possible that in FVA cells, the
Epo receptor signaling cascades already have been
activated by the Friend spleen focus-forming virus
ŽSFFV. -encoding glycoprotein gp55, which associ-
ates with the Epo receptors w46,47x. Thus, Epo-re-
sponsive genes could have already been activated in
FVA cells despite the absence of Epo in vitro culture.
Another possibility is that cytokines released from
the FVA cells in culture may induce mouse GBP3
gene in an autocrine or paracrine manner. The mech-
anism responsible for the induction of mGBP3 in
FVA cells remains to be explored. However, its
possible role in erythroid differentiation is suggested
by the high basal level of the gene expression, which
undergoes a transitory change in the course of FVA
cell differentiation in culture. By contrast, erythro-
leukemia cell lines HCD-57 and SREI-2 lacking the
capability to differentiate into hemoglobin-rich reticu-
locytes w48x have exceedingly low basal levels of
mGBP3, which remain unchanged during culture with
Epo Ždata not shown..
Mouse GBP3 transcript is also highly induced by
IFN-g in macrophages RAW264.7. This suggests
that like other GBPs, the mouse GBP3 gene product
may be involved in mediating IFN-g ’s action during
macrophage activation. It is of interest that IFNs have
been reported to inhibit normal erythropoiesis by
counteracting the proliferative effect of Epo without
affecting the ability of the hormone to induce termi-
nal differentiation w49–51x. In light of the findings
that mGBP3 is not only transiently induced during
erythropoiesis but also responsive to IFN-g , it is
tempting to suggest that mouse GBP3 gene product
may play a role in the early molecular events for the
 B.H. Han et al.r Biochimica et Biophysica Acta 1384 (1998) 373–386
initiation of the erythroid differentiation rather than
in the proliferative response to Epo.
The recombinant His-mGBP3 exhibits an intrinsic
GTPase activity that hydrolyzes GTP predominantly
to GDP Ždata not shown.. While GMP is the major
product hydrolyzed by recombinant hGBP1 w17x, GDP
rather than GMP is the predominant product of hy-
drolysis reaction catalyzed by the hGBP2 w18x and the
chicken GBP w16x. In this regard, the mouse GBP3
has a catalytic activity similar to that of the hGBP2
and of the chicken GBP. This suggests that although
there are high degrees of similarities in peptide se-
quences and in guanine nucleotides-binding activities,
there may be differences in profile of GTPase activi-
ties between members of the family of guanylate-bi-
nding-proteins.
Acknowledgements
We thank S.T. Sawyer for gifts of HCD-57 and
SREI-2 cell lines, Shivendra Shukla for FDC-P2 cell
line, Mary Kim, Jane Burnett and Elisabeth Norton
for their technical assistance. This work was sup-
ported in part by National Institute of Health Grant
AR40682 and by National Science Foundation Grant
MCB-9218686.
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