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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 54-63
A Truncated Isoform of the Human  Chain Common to the
Receptors for Granulocyte-Macrophage Colony-Stimulating Factor,
Interleukin-3 (IL-3), and IL-5 With Increased mRNA Expression
in Some Patients With Acute Leukemia
By
Rosemary E. Gale,
Robin W. Freeburn,
Asim Khwaja,
Rajesh Chopra, and
David C. Linch
From the Department of Haematology, University College London Medical
School, London, UK.
 |
ABSTRACT |
We report here a naturally occurring isoform of the human  chain
common to the receptors for granulocyte-macrophage colony-stimulating
factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (GMRC)
with a truncated intracytoplasmic tail caused by deletion of a 104-bp
exon in the membrane-proximal region of the chain. This
intracytoplasmic truncated chain (IT) has a predicted
tail of 46 amino acids, instead of 432 for C, with 23
amino acids in common with C and then a new sequence of
23 amino acids. In primary myeloid cells, IT comprised
approximately 20% of the total chain message, but was increased up
to 90% of total in blast cells from a significant proportion of
patients with acute leukemia. Specific anti-IT
antibodies demonstrated its presence in primary myeloid cells and cell
lines. Coexpression of IT converted low-affinity GMR
chains (KD 2.5 nmol/L) to higher-affinity complexes
(KD 200 pmol/L). These could bind JAK2 that was
tyrosine-phosphorylated by stimulation with GM-CSF. IT
did not support GM-CSF-induced proliferation when cotransfected with
GMR into CTLL-2 cells. Therefore, it may interfere with the
signal-transducing properties of the C chain and play a
role in the pathogenesis of leukemia.
| |
INTRODUCTION |
THE PROLIFERATION, differentiation, and
cellular functions of hematopoietic cells are regulated by the
interaction of a number of different cytokines with the receptors
expressed on the cell surface.1 Heterogeneity in the
cytokine receptor family is provided by the ligand binding-induced
oligomerization of their component chains. The erythropoietin receptor
(EpoR) and granulocyte colony-stimulating factor (G-CSF) receptor
(G-CSFR) are probably homodimers,2,3 the receptors for
granulocyte-macrophage colony-stimulating factor (GM-CSF) (GMR),
interleukin-3 (IL-3R), and IL-5 (IL-5R) are oligomers of and
chains,4 and the IL-2-R involves three different
chains.5 A common characteristic appears to be the sharing
of one "-like" receptor chain with several different
"-like" chains, for example, the specific chains for
GM-CSF, IL-3, and IL-5 all bind ligand with low affinity and complex to
form high-affinity receptors with the common chain
(C), which itself does not bind ligand.4
Similarly, gp130 can bind to the IL-6, IL-11, ciliary neurotrophic
factor, leukemia inhibitory factor, and oncostatin M receptor chains,
and the IL-2R chain binds to the IL-4, IL-7, IL-9, and IL-15
receptor chains.6
Another form of diversity reported for a number of the receptors is the
demonstration of different isoforms for a single chain. Several
isoforms of the human GMR chain have been reported from either cell
lines or normal tissues, mostly affecting the 3 end of the molecule.
Some are soluble receptors lacking the transmembrane
domain,7-9 another chain has an altered cytoplasmic
domain,10 and one isoform has an insertion just 5 of the
transmembrane domain.11 Three isoforms have also been
described affecting the 5 end of the chain. Two of them have changes
in the 5 untranslated region and would not alter the mature protein,
although they may affect translational efficiency,12 and
the other isoform completely removes the signal peptide
sequence.13 The genomic structure of the GMR chain
indicates that most of these different isoforms arise from alternative
splicing of mRNA.14 These isoforms may have different
functional consequences, as suggested by an isoform of the EpoR with a
truncated cytoplasmic region that was found to be the predominant form
in early-stage but not late-stage erythroid progenitor
cells.15 It was unable to transduce a mitogenic signal and
had a dominant-negative effect over the wild-type
receptor.16 However, only one form of the full-length human
GM-CSF/IL-3/IL-5 receptor C chain has been
reported.17
The C chain is not only required for high-affinity
binding of ligands, but is also crucial for the signaling of downstream
pathways through its intracytoplasmic tail.18 Although
there are no known kinase consensus sequences within the 432 amino acid
residues of the intracytoplasmic tail, a series of truncated mutants
have shown that it contains distinct functional
domains.18-20 In common with other receptors, the
membrane-proximal region of C appears to be both
essential and sufficient for proliferation.18,21 This
region is known to bind the family of JAK kinases, and activation of
JAK2 by tyrosine phosphorylation correlates closely with the induction
of mitogenesis.22,23 It is also essential for the induction
of c-myc, pim-1, and cis.19,24
Membrane-distal domains have been identified that are responsible for
the major tyrosine phosphorylation of proteins, induction of
c-fos and c-jun transcription, activation of the
Ras pathway, and prevention of apoptosis.18,19,25
In addition, the membrane-distal region has been associated with the
induction of differentiation20 and negative regulation of
receptor signals.26
We describe here an isoform of the C chain with deletion
of a 104-bp exon just 3 of the transmembrane region. This resulted in
a truncated receptor chain with an intracytoplasmic tail containing 23
amino acids in common with C and then an altered
C-terminal tail of a further 23 amino acids. JAK2 bound to this isoform
and was tyrosine-phosphorylated by stimulation with GM-CSF; however, it
did not transduce a mitogenic signal when transfected into CTLL-2
cells. In primary myeloid cells and myeloid cell lines, transcript and
protein levels comprised approximately 10% to 25% of the total
chain. Increased transcript levels were detected in blast cells from
approximately 75% of patients with acute myeloid leukemia (AML),
suggesting that this isoform may play a role in the pathogenesis of the
disease.
| |
MATERIALS AND METHODS |
Samples and cell culture.
The hematopoietic cell lines TF-1,27 HL60,28
U937,29 and CTLL-230 were grown in RPMI 1640
supplemented with 10% fetal calf serum (FCS), plus 5 ng/mL GM-CSF for
TF-1 cells and 30 U/mL IL-2 for CTLL cells. Neutrophils from normal
healthy volunteers, mononuclear cells from normal human bone marrow,
and leukemic blast cells from patients at presentation with AML and
acute lymphoblastic leukemia (ALL) were all prepared by
density-gradient centrifugation (Nycomed, Oslo, Norway).
CD34+ cells were prepared using a Ceprate SC column
(CellPro, Bothell, WA) and were 90% pure. Blast cells
(50 × 106) from AML patients were cultured for 9 or 10
days in Iscove's medium supplemented with 20% FCS, 10 ng/mL IL-3, 10
ng/mL GM-CSF, and 100 ng/mL G-CSF. Cytospins were prepared before and
after culture, and morphology was examined using
May-Grünwald-Giemsa staining.
RNA preparation.
Total cellular RNA was extracted using a standard method of guanidinium
isothiocyanate lysis and ultracentrifugation through cesium
chloride.31
Reverse transcriptase-polymerase chain reaction.
One microgram of total RNA was reverse-transcribed using 250 ng
oligo-dT as a primer (Promega, Madison, WI) in a total volume of 20
µL containing 1× Taq polymerase buffer, 5.25 mmol/L
MgCl2, 1 mmol/L each dNTP, 20 U RNAse inhibitor, and 3 to 5
U AMV reverse transcriptase (RT; Promega). Reactions were incubated at
42°C for 1 hour and then at 95°C for 5 minutes. A 4-µL RT
reaction was used for polymerase chain reaction (PCR) in a total volume
of 20 µL containing 1× Taq polymerase buffer, 2.25 mmol/L
MgCl2, and 40 ng of each primer designed to amplify a
fragment of 339 bp between nucleotides 1281 and 1620 flanking the
transmembrane region of the C chain (Table 1, primers 1
and 2). This mixture was heated at 95°C for 5 minutes and held at
85°C while 1 U Taq polymerase was added, and then 35 cycles of 95°C
for 30 seconds, 64°C for 30 seconds, and 72°C for 1 minute were
performed. The final extension step was 72°C for 5
minutes.
For longer-length RT-PCR, 1 to 2 µg total RNA from TF-1 cells was
heated at 65°C for 5 minutes with 250 ng oligo-dT, cooled at room
temperature for 10 minutes, and then reverse-transcribed in a total
volume of 20 µL containing 2 µL 10× Stratascript buffer, 10
mmol/L DTT, 1 mmol/L of each dNTP, 20 U RNAse inhibitor, and 50 U
Stratascript RNAse H minus reverse transcriptase (Stratagene, La Jolla,
CA). Reactions were incubated at 37°C for 1 hour and then at 90°C
for 5 minutes. PCR was performed on a 4-µL RT reaction as described
earlier using primers 2 and 10 (Table 1), which cover the extracellular
portion, transmembrane region, and membrane-proximal intracytoplasmic
tail of the chain, and the extension time was 2 minutes at 72°C
for each cycle. The PCR product was electrophoresed through 1%
low-melting-point agarose, the fragment around 1.5 to 1.6 kb was cut
out and melted, and 5 µL was placed in a fresh 50-µL PCR
using primers 1 and 2 (Table 1).
Semiquantitative RT-PCR using primers 1 and 2 was performed as
described earlier except that an [-32P] end-labeled
primer was incorporated and the number of cycles was reduced to 25. PCR
products were separated through 6% polyacrylamide (19:1
acrylamide:bis-acrylamide) in 1× TBE, and the gels were dried and
exposed to Hyperfilm-MP (Amersham, Bucks, UK). Signals were quantified
using a Hoefer densitometer (Hoefer Scientific Instruments, San
Francisco, CA).
Genomic DNA PCR and sequencing.
One microgram of genomic DNA prepared from TF-1 cells was used as
template in a 100-µL hot-start PCR as described earlier except that
the extension time at 72°C was lengthened to 3 minutes and
combinations of primers 1 to 9 (Table 1) were used. Products were
sequenced using either the chain-termination method with modified T7
DNA polymerase (Sequenase Version 2.0; Amersham, Bucks, UK) or the
fmol DNA sequencing kit with kinase-labeled primers (Promega).
RNAse protection assays.
For RNA probes, a 339-bp PCR product was prepared from the KH97 GMR
chain cDNA clone kindly provided by Dr Miyajima17 using
primers 1 and 2 (Table 1), ie, nucleotides 1281 to 1620, and subcloned
into the pGEM-T vector (Promega). Rsa I digestion of a clone
with the insert in the reverse orientation produced a 1370-bp fragment
containing the T7 RNA polymerase promoter and GMR sequence from
nucleotides 1429 to 1620. Antisense probe was synthesized in a total
volume of 20 µL containing 500 ng template, 1× transcription buffer
(Promega), 10 mmol/L DTT, 500 µmol/L each for ATP, GTP, and TTP, 50
µCi [-32P]CTP (>400 Ci/mmol; Amersham), 20 to 40 U
RNAse inhibitor, and 10 to 20 U T7 RNA polymerase (Promega). The
mixture was incubated at 37°C for 90 minutes, and then 5 U RQ DNAse 1
(Promega) was added and incubation continued for 30 minutes. The
reaction was phenol/chloroform-extracted, ethanol-precipitated, and
dissolved in 100 µL TES (10 mmol/L Tris, pH 7.5, 1 µmol/L EDTA, and
0.1% sodium dodecyl sulfate [SDS]). Sense probe was similarly
transcribed using T7 polymerase from a 1,465-bp fragment obtained by
Dde I digestion of a clone containing the 339-bp insert in the
forward orientation.
For the protection assays, between 10 and 25 µg total cellular RNA
was freeze-dried and resuspended in 25 µL hybridization buffer (80%
deionized formamide, 40 mmol/L PIPES, pH 6.4, 1 mmol/L EDTA, 400 mmol/L
NaCl, and 0.1% SDS). Labeled probe (0.5 to 1 µL) was added, and the
mixture was heated at 90°C for 10 minutes and incubated at 50°C
overnight. Samples were nucleased in 10 mmol/L Tris, pH 7.5, 5 mmol/L
EDTA, 300 mmol/L NaCl, 14 µg RNAse A, and 3,000 U RNAse T1
(Boehringer Mannheim, Lewes, UK) for 60 minutes at 30°C and then
incubated with 100 µg proteinase K (Boehringer) and 10 µL 20% SDS
for 30 minutes at 37°C. The reactions were extracted with
phenol/chloroform, and the RNA was precipitated with ethanol, washed
with 70% ethanol, and resuspended in loading buffer. Protected
fragments were resolved on 6% denaturing polyacrylamide gels (19:1
acrylamide:bis-acrylamide, 7 mmol/L urea) in 0.5× TBE. The gels were
dried and exposed either to preflashed XAR-5 film (Kodak, Rochester,
NY) at  80°C for 1 to 5 days or to phosphorimager plates and
evaluated on a Fujimax bas 1000 phosphorimager (Fuji Photo Film Co,
Tokyo, Japan). The signals were quantified using
Millipore Whole Band Analyzer software (Millipore,
Watford, UK) on a Sun Sparc workstation and corrected for GTP content
to obtain the relative percentage of the different isoforms.
Construction of GMRIT clone.
One microgram of RNA from TF-1 cells was reverse-transcribed as
described earlier, and PCR was performed using primers 1 and 9 (Table
1) to amplify fragments of 529 bp (from the C chain) and
425 bp (IT) covering nucleotides 1281 to 1810 of the
published GMR sequence.17 DNA from the KH97 GMR clone
and the 425-bp PCR fragment were both digested with BssHII and
BglII, restriction enzymes that cut uniquely at nucleotides
1319 and 1705 of the GMRC, respectively, producing
fragments of 5,600 plus 386 bp for the clone and 282 bp for the PCR
product. The 5,600-bp and 282-bp fragments were extracted from
low-melting-point agarose, ligated using T4 DNA ligase (Promega),
electroporated into JM109 bacteria, and selected on ampicillin.
Positive clones were sequenced across the entire region inserted from
the PCR fragment to check for possible Taq errors.
COS-7 cell transfection.
Approximately 8 × 106 COS-7 monkey cells grown to
semiconfluence in Dulbecco's modified medium with sodium pyruvate,
1,000 mg/L glucose, and 10% FCS were electroporated with either water,
10 µg GMR + 10 µg GMRC DNA (C),
or 10 µg GMR + 10 µg GMRIT DNA
(IT) using a Gene Pulser (BioRad, Hercules, CA) with
capacitance 500 µFD, 0.4 kV, and then cultured for 72 hours in Costar
plates or petri dishes. The GMR construct was obtained by RT-PCR of
TF-1 RNA using experimental conditions as described earlier with
primers designed to amplify the full-length cDNA sequence32
(5-GTAGAACCCTGTACGTGCTT-3 and 5-AGAAAACAGTTCCCCCGTGT-3) and an
annealing temperature of 62°C. The PCR product was ligated into the
vector pCR1000 (Invitrogen, San Diego, CA) and then
subcloned into the expression vector pSVL (Pharmacia, St Albans, UK) by
blunt-ending the HindIII/SacII fragment with T4 DNA
polymerase.
CTLL cell transfection.
For transfection into CTLL cells, the GMR, C, and
IT chains were subcloned into the vector pcDNA3
(Invitrogen) and linearized using Sca I digestion.
Approximately 8 × 106 CTLL cells were transfected with
either 25 µg vector DNA (V), 25 µg GMR DNA (), 25 µg GMR
+ 25 µg GMRC DNA (C ), or 25 µg
GMR + 25 µg GMRIT DNA (IT) by
electroporation at 960 µFD, 0.35 kV and then selected in 1 mg/mL G418
(GIBCO-BRL, Paisley, Scotland). Expression of transcripts from the
transfected GMR constructs was checked by RT-PCR. Individual clones
were plucked by plating 250 transfectants/mL in Methocult (Terry Fox
Laboratories, Metachem, UK) supplemented with G418 and IL-2.
Binding studies.
[125I]GM-CSF (human recombinant Escherichia coli
product) with specific activity of 850 to 1,200 Ci/mmol was obtained
from Amersham, and the specific activity was confirmed by analysis of
maximal binding capacity and self-displacement assay. Binding was
performed in situ in 12- or 24-well Costar plates. Two to 3 ×
105 cells were incubated with varying concentrations of
125I-GM-CSF in binding buffer (RPMI 1640, 25 mmol/L HEPES,
and 2% FCS, pH 7.4) for 2 hours at 37°C. Parallel samples were
incubated with greater than a 100-fold molar excess of unlabeled GM-CSF
to control for nonspecific binding. After incubation, samples were
washed four times in ice-cold binding buffer and then lysed with 1
mmol/L NaOH and counted. Equilibrium binding data were analyzed using
the LIGAND program (Biosoft, Cambridge, UK).
Surface receptor expression.
Aliquots of 1 × 106 CTLL cells in 50 µL binding buffer
were incubated in round-bottom 96-well plates with monoclonal mouse
anti-human antibodies to the GMR chain (S20; Santa Cruz
Biotechnology, Santa Cruz, CA) or extracellular GMR chain (DC-9;
kindly provided by Dr J. Tavernier, Gent, Belgium) at a final
concentration of 2.5 µg/mL in 3% BSA and 0.1% sodium azide for 1
hour on ice. The cells were then washed three times in binding buffer
at 4°C and incubated with 50 µL [125I]-labeled sheep
anti-mouse F(ab)2 fragments (0.3 µg/mL in binding
buffer; specific activity, 500 to 2,000 Ci/mmol; Amersham) for 30
minutes on ice. Cell-associated radioactivity was separated from
unbound antibody by centrifuging through chilled FCS, and then these
samples were snap-frozen and counted.
Preparation of anti-IT antibodies.
Rabbit polyclonal antipeptide antibodies were raised against the novel
C-terminal tail of the IT chain and purified by caprylic
acid precipitation and absorption on a peptide-specific
column.33
Immunoprecipitation, SDS-PAGE, and Western blotting.
For cell lysates, transfected COS-7 cells were harvested with a rubber
policeman in 0.5 mL lysis buffer (137 mmol/L NaC1, 20 mmol/L Tris, pH
8, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 1% NP-40,
10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L
-glycerophosphate, 2 mmol/L EDTA, 1 mmol/L PMSF, and 10 µg/mL each
of aprotinin, leupeptin, and pepstatin), and then an equal volume of
2× Laemmli sample buffer was added to the supernatant and the sample
was boiled for 10 minutes. For immunoprecipitates, transfected COS-7
cells were incubated with or without 100 ng/mL GM-CSF
(Behringwerke, Marburg, Germany) for 5 minutes at 37°C, lysed in situ
with 0.5 to 1 mL ice-cold lysis buffer, and then harvested and
incubated for 30 minutes on ice. After centrifugation at
12,000g for 10 minutes, the supernatant was incubated with
either JAK2 polyclonal antiserum (UBI, Lake Placid, NY), the anti-
antibody DC-9, or the anti-IT antibody. After 2 to 14
hours, 60 µL protein A-agarose (50% in lysis buffer; Repligen,
Cambridge, MA) was added, incubated end-over-end for 2 hours, washed
four times in lysis buffer, and then resuspended in 50 µL 2×
Laemmli sample buffer and boiled for 10 minutes. For immunoprecipitates
of cell lines or primary myeloid cells, aliquots of cells were
suspended in ice-cold lysis buffer at 50 × 106 mL and
processed as already described. Proteins were electrophoresed through
SDS-7% polyacrylamide gels and transferred to nitrocellulose membrane
(Hybond-C Extra; Amersham). The blots were incubated in PBS, pH 7.4,
containing 3% BSA (Fraction V; Sigma, St Louis, MO) to block
nonspecific binding sites and then incubated with the appropriate
antibody, JAK2 (1 in 1,000), 4G10 antiphosphotyrosine antibody (1
µg/mL; UBI), DC-9 (2 µg/mL), or anti-IT, for 2 hours
at room temperature. After washing four times in PBS/0.05% Tween-20,
pH 7.4, blots were incubated with peroxidase-conjugated secondary
antibody (Dakopatts, High Wycombe, UK) for 2 hours at room temperature,
washed four times in PBS/0.05% Tween-20, pH 7.4, and then developed by
enhanced chemiluminescence (Amersham) and analyzed by autoradiography.
[3H]thymidine assay.
Aliquots of CTLL clones were washed four times and then incubated with
IL-2 or varying concentrations of GM-CSF in triplicate at 5 ×
104 cells/well for 72 hours. [3H]thymidine
(0.5 µCi/well, 26 Ci/mmol; Amersham) was added and the incubation
continued for a further 4 hours before the cells were harvested on
fiberglass filters and counted.
| |
RESULTS |
Identification of IT.
RT-PCR of RNA from the TF-1 cell line using primers 1 and 2 (Table 1)
consistently yielded two products, one of 339 bp, as expected from the
published sequence for the GMR chain,17 and another of
approximately 240 bp. The relative proportion of this smaller fragment
varied with the sample analyzed: for TF-1, HL60, and U937 cell lines
and primary neutrophils, it comprised approximately 10% to 25% of the
total product as estimated visually, but was increased to greater than
50% of total in RNA from leukemic blasts of some patients with AML and
ALL (Fig 1). Preparative PCR, purification,
and sequencing of the fragment showed that there was a deletion of 104
bp between nucleotides 1491 or 1492 and 1595 or 1596. This is just 69
bp 3 of the end of the transmembrane region in the membrane-proximal
region of the intracytoplasmic tail. The deletion causes a frameshift
alteration and introduces a premature stop codon, which if translated
into protein would produce a chain with a truncated
intracytoplasmic tail of just 46 amino acids instead of 432, hence the
designation IT for intracytoplasmic truncated (Fig
2A). Twenty-three amino acids of the
IT tail would be identical to the C chain
followed by a new sequence of 23 amino acids (Fig 2B). Other bands were
sometimes seen after PCR amplification (Fig 1), but they were always
minor components of the total product and were not investigated
further.
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| Fig 1.
RT-PCR analysis of the GMRC chain between
nucleotides 1281 and 1620 of the published sequence17 using
RNA from hematopoietic cell lines (TF-1 and U937) and leukemic blasts
of 2 patients with AML (Pt). L, ladder. Primers 1 and 2 were used
(Table 1), and the expected fragment size was 339 bp.
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| Fig 2.
(A) Protein structure of the GMRC chain
compared with predicted structure of the IT chain formed
by splicing out the 104-bp exon in the membrane-proximal region. ( )
New carboxy-terminal tail of 23 amino acids created by the deletion.
(B) Amino acid sequence of the intracytoplasmic tails of
C and IT chains. The point of sequence
divergence is marked by an arrow.
|
|
To show that full-length transcripts of the truncated chain isoform
could be obtained, TF-1 RNA was reverse-transcribed using oligo-dT as a
primer with Stratascript RNAse H minus reverse transcriptase
(Stratagene), and PCR was performed with a primer located at the 5 end
of the extracellular portion of the published sequence (Table 1, primer
10) and primer 2, which is just 3 of the deletion. A band of
approximately 1,600 bp, as expected, for the full-length
C was observed plus a slightly smaller band. The bands
were cut out from a low-melting-point agarose gel as one fragment and
used in a new PCR performed with primers 1 and 2 (Table 1). Both the
C band (339 bp) and IT band (235 bp) were
seen in approximately the same proportion as originally observed,
indicating that full-length transcripts of the IT chain
were present in the RNA.
The IT isoform arises from the
alternative splicing out of an exon.
PCR of genomic DNA from TF-1 cells and direct sequencing of the
products showed that the 104 bp deleted in the IT
isoform was a complete exon, with an intron of approximately 850 bp
between nucleotides 1492/1493 and another intron of approximately 750
bp between nucleotides 1596/1597 (Table 2).Two other introns in the transmembrane region could also be
demonstrated between nucleotides 1343/1344 (~1,400 bp) and 1434/1435
(187 bp) (Table 2). Further PCR and Southern blot analysis of genomic
DNA showed that the remainder of the intracytoplasmic tail, nucleotides
1597 to at least the Xba I restriction site at 2991, was
contained within one exon (data not shown).
Quantification of IT mRNA.
The relative levels of IT and C
transcripts present in hematopoietic cell lines and primary cells were
quantified using two methods. To show that the deleted fragment was not
an artifact produced by secondary structure of the RNA, RNAse
protections were first performed on total cellular RNA from cell lines,
primary neutrophils, normal bone marrow, purified CD34+
cells, and leukemic blasts. The RNA probe of 248 bp was designed to
protect a 191-bp fragment from the C chain (nucleotides
1429 to 1620) and two fragments of 64 bp (nucleotides 1429 to 1493) and
24-bp (nucleotides 1596 to 1620) from the IT chain.
Typical results obtained are shown in Fig 3. Secondly, semiquantitative
RT-PCR using an end-labeled primer was performed on some RNA samples.
There was good agreement between results
obtained using the two methods. Quantification of IT
showed that it comprised between 10% and 25% of the total chain
message for U937 and TF-1 cells, similar to the levels obtained for
primary myeloid cells, normal bone marrow (14.6% ± 6.9%,
n = 7), purified neutrophils (13.8% ± 4.5%, n = 6), and
CD34+ cells (16.6% ± 8.4%, n = 4). However,
IT levels varied from 10% to 90% in blast cells from
patients with AML and ALL and was greater than 80% of the total
message in 24 of 32 (75%) AML patients and two of 11 (18%) ALL
patients (Fig 4). The one ALL patient with a IT level of
91% had an immunophenotype consistent with common ALL (CD10 68%, CD19
97%, peroxidase-negative, TdT-positive, CD33 2%, CD13 14%).
There was no correlation between the
IT expression detected and arrest of leukemic cells at
different stages of differentiation as defined by the
French-American-British classification.
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| Fig 3.
RNAse protection analysis of the GMR chain using total
RNA from hematopoietic cell lines (TF-1), primary myeloid cells
(purified neutrophils, CD34+ cells, and mononuclear cells
from normal bone marrow), and leukemic blasts from patients with AML.
The full-length probe of 248 bp protected a fragment of 191 bp from
nucleotides 1429 to 1620 of the published C chain
sequence17 and 64 bp for the IT chain.
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| Fig 4.
Relative expression of IT mRNA as detected
by RNAse protection assays ( ) or semiquantitative RT-PCR ( ) on
total RNA from hematopoietic cell lines, primary myeloid cells, and
leukemic blasts of patients with ALL or AML of varying FAB types (M0 to
M6).
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Relationship of IT transcript levels to
differentiation.
To investigate whether the level of IT transcripts
changed with differentiation of myeloid cells, leukemic blasts from six
AML patients were cultured in liquid suspension in a cocktail
containing IL-3, GM-CSF, and G-CSF. RNA was prepared at days 0 and 9 or
10, and semiquantitative RT-PCR was performed. Morphologic
differentiation was observed in each patient, and the relative
proportion of IT message decreased by approximately 10%
to 30% (Table 3). The difference in IT expression
before and after differentiation was highly significant
(P = .0009, paired t
test).
IT can form a high-affinity GM-CSF
receptor.
To analyze GM-CSF binding to the truncated chain, IT DNA
was transfected with GMR DNA into COS-7 cells. Two classes of
receptor affinity were obtained with KD values of 2.5
nmol/L, consistent with reported values for the GMR
chain,32 and 200 pmol/L (Fig 5). This indicated that the
IT could be expressed as protein and was able to convert
low-affinity chains to a higher-affinity complex with
binding affinity similar to that reported
for the full-length C chain.17
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| Fig 5.
Scatchard analysis of 125I-GM-CSF binding to
COS-7 cells transiently transfected with either GMR () or GMR
+ IT ().
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JAK2 binds to IT and is phosphorylated by
GM-CSF.
Previous studies have shown that the tyrosine kinase JAK2 binds to the
C chain, and its activation requires the
membrane-proximal region.23 To examine whether JAK2 could
associate with IT, COS-7 cells were transfected with
GMR and either C or IT and grown to
semiconfluence. The cells were then stimulated with or without GM-CSF,
harvested, and immunoprecipitated with an anti-JAK2
antibody. Immunoblotting with the anti-
antibody DC-9 demonstrated that the IT chains
coimmunoprecipitated with JAK2 (Fig 6A). Blotting with the
phosphotyrosine antibody 4G10 showed that JAK2 associated with
IT could be phosphorylated on tyrosine residues by
stimulation of the cells with GM-CSF (Fig 6B).
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| Fig 6.
JAK2 association with the IT chain and
subsequent phosphorylation by stimulation with GM-CSF. (A) Cell lysates
from COS-7 cells transfected with either water or GMR +
IT DNA (IT) were immunoprecipitated
(IP) with an anti-JAK2 antibody, and the proteins were separated on
SDS-PAGE, transferred to nitrocellulose, and probed using antibodies to
the extracellular portion of the chain (DC-9) or JAK2. (B) COS-7
cells transfected with either GMR + C
(C) or GMR + IT
(IT) were stimulated without () or with (+)
saturating concentrations of GM-CSF, lysed, and immunoprecipitated with
an anti-JAK2 antibody. After SDS-PAGE and transfer to nitrocellulose,
the blots were probed with an antiphosphotyrosine antibody (4G10) or
anti-JAK2 antibody.
|
|
Protein expression of the IT chain.
Anti- immunoprecipitation and Western blotting of transfected COS-7
cells yielded a protein with molecular mass of approximately 70 kD for
IT, in comparison to 130 kD for C (Fig
7A). A degradation product of variable intensity was consistently
observed at approximately 80 kD in COS-7 cells transfected with
C. Similarly, anti-
immunoprecipitation and Western blotting of TF-1 cells yielded a
protein band at the expected position for IT, but it was
always present at a relatively low level and was often obscured by the
C degradation product (Fig 7C). Polyclonal antibodies
were therefore raised to the novel C-terminus of the IT
chain. Using cell lysates from transfected COS-7 cells, the antibody
was shown to be specific for IT chains (Fig 7B). It did
not cross-react with C and could be competed out with
specific peptide but not an unrelated peptide derived from the
retinoblastoma protein. Anti-IT immunoprecipitates of
TF-1 cells and purified CD34+ cells cultured for 8 days
with stem cell factor, IL-3, and IL-6 were probed with the anti-
antibody and showed that IT was expressed as protein in
myeloid cell lines and primary myeloid cells but was absent from the
murine T-cell line, CTLL (Fig 7C). At least two bands were always
observed for the IT chain using either anti- or
anti-IT antibodies. These were thought to be due to
variable glycosylation of the chain, as only one smaller band was
observed in transfected COS-7 cells incubated with tunicamycin (data
not shown).
View larger version (22K):
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[in a new window]
| Fig 7.
Protein expression of the IT chain. (A)
Anti- immunoprecipitation and Western blotting of COS-7 cells
transiently transfected with either C or
IT. (B) Western blots of cell lysates from COS-7 cells
transfected with C or IT and probed with
either anti- antibody, anti-IT antibody, or
anti-IT + specific or unrelated peptide. (C) Western
blots of anti- or anti-IT immunoprecipitates from
hematopoietic cell lines (TF-1 and CTLL) or primary CD34+
cells cultured for 8 days in stem cell factor, IL-3, and IL-6 and
probed with anti- antibody.
|
|
The IT chain does not transduce a
mitogenic signal.
To examine the ability of IT to support a proliferative
signal, CTLL-2 cells were stably transfected with either vector (V),
GMR (), GMR + GMRC (C), or
GMR + GMRIT (IT). CTLL-2 cells were
chosen because, unlike the more commonly used BA/F3 cells, they do not
express endogenous C chains that could interfere with
the signal obtained and were dependent on IL-2, not IL-3. Individual
clones were selected, and expression of the different chains was
confirmed by both surface-antibody binding and Western blotting of
immunoprecipitates. Proliferation in response to varying concentrations
of GM-CSF was examined using [3H]thymidine uptake and
compared with the response to IL-2. Two C and
IT clones were examined, and the assays were repeated
at least twice. The clones containing vector, GMR, or
IT did not show any proliferation in the presence of
GM-CSF, whereas the two C clones showed a
proliferative dose-response effect (Fig 8).
View larger version (10K):
[in this window]
[in a new window]
| Fig 8.
[3H]thymidine uptake assay of CTLL-2 clones
transfected with vector (), GMR (), GMR +
C (C), or GMR + IT
(IT) and stimulated with increasing concentrations of
GM-CSF. Results are expressed as a percentage of the IL-2 growth for
each clone.
|
|
| |
DISCUSSION |
The human C chain common to the GM-CSF/IL-3/IL-5
receptors is located at chromosome 22q12.2  q13.1,34 and
unlike the mouse, where two distinct but highly homologous genes have
been found for the subunits, AIC2A and AIC2B, 35 only
one human gene has been found.17 A number of different
cDNAs with insertions and/or deletions were isolated in the
original cloning of C from TF-1 cells17 and
were thought to be created by alternative splicing rather than encoded
by a distinct gene, since their alterations were found at sites
corresponding to the exon-intron junctions of the mouse AIC2
genes.35 We have found that full-length transcripts
containing a deletion identified in one of these alternative forms,
clone KH85, can be detected as approximately 10% to 25% of the total
chain message in hematopoietic cell lines and primary hematopoietic
cells and up to 90% of the mRNA in blast cells from patients with
acute leukemia (Fig 4). We have called this isoform
intracytoplasmic truncated (IT) because the 104-bp
deletion just 3 of the intracytoplasmic tail causes a frameshift
alteration with a premature stop codon, potentially producing a protein
with a truncated intracytoplasmic tail of just 46 amino acids instead
of 432. The finding that IT decreased when AML blasts
were cultured with IL-3, GM-CSF, and G-CSF (Table 3) raises the
possibility that its expression is differentiation stage-specific in
the leukemic cells. However, expression of IT was not
related to AML FAB type, with high levels found in all three cases of
M3 leukemia examined (Fig 4). Furthermore, increased levels of
IT expression were not found in purified normal
CD34+ cells (mean, 16.6% ± 8.4%, n = 4). It is
possible that a primitive subpopulation of CD34+ cells
express high levels of IT mRNA, but even if this is the
case, the presence of high levels in leukemic samples showing myeloid
differentiation is indicative of an aberrant phenotype. The finding of
a very high IT level in one ALL patient sample indicates
that this is not a myeloid-specific phenomenon.
Sequencing of genomic DNA demonstrated that the IT chain
had arisen from the splicing out of a complete exon, nucleotides 1493
to 1596 of the original cDNA sequence,17 with the
appropriate donor and acceptor splice sites present (Table 2). The
genomic structure in this region is similar to that reported for the
mouse subunit genes AIC2A and B.35 With the exception
of the first 5 nucleotide in the human gene, G, the transmembrane
domain is encoded in one exon of 91 bp, exon 11 in the mouse genes.
This is followed by two small exons of 58 and 104 bp, respectively, and
then a large exon that includes most of the intracytoplasmic tail
sequence. This genomic organization fits a common pattern found in
several cytokine receptor chains, indicating that there may be a common
evolution of growth factor receptor chains, possibly a shared ancestral
gene.14,36
Although the protein could be expressed in COS-7 cells and was able to
convert low-affinity binding GMR chains to a higher-affinity GM-CSF
binding protein, we were unable to evaluate protein levels in
hematopoietic cells by immunoprecipitation and blotting with the
anti- antibody because of an 80- to 90-kD degradation product from
the C chain (Fig 7). In primary cells, this was
consistently observed as a broad band, probably due to variable
glycosylation of the chain, which interfered with detection of the
IT band. However, using specific anti-IT
antibodies, we were able to demonstrate that full-length
IT chains are present both in the TF-1 erythroleukemia
cell line and in primary myeloid cells. We were consistently unable to
detect either C or IT in blast cells from
patients with AML using the anti- or -IT antibodies,
presumably because these cells often express less than 100
high-affinity GMR/cell,37,38 in comparison to 2,000 ± 450
and 1,100 ± 200 for TF-1 cells and neutrophils,
respectively.39
The first 23 amino acids of the intracytoplasmic tail of the
IT chain that it has in common with the published
C include the sequence known as box 1, a highly
conserved sequence of amino acids including the proline-X-proline found
in human gp130, G-CSFR, IL-2R, EpoR, IL-7R, and IL-4R
chains.21 This sequence is necessary for binding and
subsequent phosphorylation of the tyrosine kinase
JAK2.40,41 Consistent with this, JAK2 could bind to the
IT isoform and was phosphorylated on tyrosine residues
by GM-CSF stimulation (Fig 6). However, although box 1 is essential for
induction of a mitogenic signal,42 JAK2 phosphorylation
alone was insufficient to support a proliferative signal, as indicated
by the lack of response to GM-CSF in CTLL clones transfected with
GMR and IT (Fig 8). A second region of homology
called box 2, which is found in a number of receptors including human
C and is known to be important for signal transduction
in gp130, is not present in IT.21 This
region is not required for JAK2 association and phosphorylation,
although it may play a role in enhancing
proliferation.40-42 The new sequence of 23 amino acids
created by the frameshift contained two prolines, two cysteines, a
serine, and a threonine (Fig 2B). There are no known consensus binding
sequences within this new sequence.
Although the IT chain does not support proliferation, it
could modulate C function, particularly if the
chains dimerize in the receptor complex.43,44 The effect
would be especially noticeable in leukemic blast cells, where it may be
the major species of receptor chain. It might act as a
dominant-negative for proliferation, as reported for a truncation
mutant of the EpoR,16 or for differentiation, as suggested
for truncation mutants of the G-CSFR.45,46 It is noteworthy
that IT expression does not prevent in vitro
differentiation of myeloid leukemic blast cells, although subtle
modifications of differentiation would not be detected in such systems.
It remains possible that high levels of IT expression
contribute to the differentiation arrest seen in AML.
| |
FOOTNOTES |
Submitted April 22, 1997;
accepted August 22, 1997.
Address reprint requests to Rosemary E. Gale, PhD, Department of
Haematology, University College London Medical School, 98
Chenies Mews, London WC1E 6HX, UK.
Supported by the Kay Kendall Leukaemia Fund (R.E.G. and R.W.F.), the
Medical Research Council of Great Britain (A.K. and R.C.), and the
Wellcome Trust (A.K.).
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr S. Devereux for the GMR construct, and S.
Langabeer for some of the RNA samples.
| |
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Abstract
Introduction
Abstract