From the Hanson Centre for Cancer Research and Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The high affinity receptor for human
granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a
cytokine-specific GM-CSF1 is a potent
cytokine that promotes the survival, proliferation, differentiation,
and functional activity of a wide variety of hemopoietic cell types
including monocytes/macrophages, granulocytes, and myeloid progenitor
cells (reviewed in Ref. 1). Like other cytokines, GM-CSF exerts its
biological activities through binding to specific receptors on the
surface of target cells. The high affinity receptor for human GM-CSF
(hGMR) is composed of a cytokine-specific Although the stoichiometry of subunits in active hGMR, hIL-3R, and
hIL-5R complexes remains unresolved, it has become clear that
ligand-induced The isolation of constitutively active cytokine receptor mutants has
provided a useful tool for examining the normal activation process of
some receptors (e.g. erythropoietin receptor and c-Mpl (14,
15)), since these mutant receptors most likely mimic the structure of
the normal cytokine-activated receptors. With regard to the
GMR/IL-3R/IL-5R system, we have previously combined random mutagenesis
with retroviral expression cloning to identify constitutively
activating point mutations in h One possible explanation for the cell type specificity of the I374N
mutant is that a molecule that is present in FDC-P1 (and other myeloid)
cells is required for its constitutive activity. We report here the use
of retroviral expression cloning to identify the mouse GMR Cell Lines--
BOSC 23 (20) and Construction of the FDC-P1 cDNA Library--
cDNA
library construction was performed essentially as described by Rayner
and Gonda (25). Briefly, cDNA was synthesized from the mouse
IL-3/GM-CSF-dependent myeloid cell line FDC-P1 (26) and
size-selected for cDNA fragments greater than 500 base pairs.
Following digestion with BamHI and XhoI, the
size-selected cDNA was ligated directionally into the pRUFNeo
retroviral expression vector (25). The library was amplified in
Escherichia coli by electroporation of aliquots of the
ligated FDC-P1 cDNA. The resultant colonies from each
electroporation were harvested, and plasmid DNA was prepared from each pool.
Infection of Target Cells with the FDC-P1 cDNA
Library--
Retroviral DNA was used to generate a library of
retroviruses by a modification of the method described by Rayner and
Gonda (25). Briefly, amphotropic BING packaging cells were transiently transfected using the procedure described by Jenkins et al.
(27) with 10 µg of retroviral plasmid per 60-mm culture dish (seeded 18 h previously with 2 × 106 cells). At 48 h post-transfection, virus-containing supernatants were filtered and
used to infect ecotropic PCR Recovery and Sequencing of cDNAs from Factor-independent
Cells--
PCR was performed on 100 ng of genomic DNA (prepared
essentially as described by Hughes et al. (28)) with an XL
PCR kit (Perkin-Elmer) under conditions recommended by the
manufacturer. The primers used for amplification were RCF1 (25), which
corresponds to the vector gag sequence approximately 80 base
pairs 5' of the polylinker in the pRUFNeo vector and RCR2
(5'-ATAGCCTCTCCACCCAAGCG-3'), which corresponds to the
MC1Neo sequence 364 base pairs 3' of the polylinker. PCR
products were agarose gel-purified, and the 5'- and 3'-ends were
sequenced with PCR primers. Internal primers corresponding to cDNA
sequences obtained from initial sequencing with PCR primers were
subsequently used to fully sequence PCR products. Sequencing reactions
were performed using a Taq DyeDeoxy Terminator Cycle
Sequencing kit (Perkin-Elmer), and sequence data were obtained by
running reactions on an ABI Prism 377 DNA Sequencer.
Receptor Expression Constructs--
The pRUFNeo/mGMR
To introduce the 8-amino acid DYKDDDDK FLAG polypeptide (Eastman Kodak
Co.) at the N terminus of mGMR
The HSV-derived 11-amino acid QPELAPEDPED polypeptide (Novagen) was
inserted after the signal sequence of the wild-type and I374N mutant
The following GMR
Extracellular truncations of mGMR
The cytoplasmic truncation mutant of mGMR
All PCRs were performed on 20 ng of plasmid DNA with Pfu DNA
polymerase (Stratagene) under conditions recommended by the
manufacturer. The structures of all mutated or chimeric cDNAs were
verified by sequencing.
Infection of Hemopoietic Cells--
Retroviral infection of
mouse BAF-B03 cells and CTL-EN cells was performed using either stably
transfected
Retroviral infection of human UT7 cells was performed using amphotropic
BING packaging cells based on the method for infecting mouse
hemopoietic cells with BOSC 23-derived retroviruses (27). Briefly, BING
cells were transiently transfected with 10 µg of retroviral DNA,
following which infections were performed by co-cultivating 3 × 105 UT7 cells with the BING cells for 48 h in growth
medium supplemented with 4 µg/ml polybrene. Cells were harvested and
selected in liquid culture medium containing growth factor and G418 at
1.5 mg/ml.
Analysis of Receptor Subunit Expression by Flow
Cytometry--
Expression of receptor subunits on the surface of
infected cells was detected by high sensitivity immunofluorescence
followed by flow cytometry on an Epics-Profile II analyzer (Coulter).
High sensitivity immunofluorescence was performed by incubating cells with primary antibody followed by biotinylated anti-mouse IgG (Vector
Laboratories) and streptavidin-phycoerythrin (Caltag Laboratories). Expression of FLAG epitope-tagged mGMR Cell Proliferation Assays--
Infected cells were washed twice,
and triplicate samples of equal cell number (5 × 103)
were cultured in a 96-well microtiter plate with or without appropriate
growth factor for 72 h. Cell proliferation was measured by the
CellTiter 96 nonradioactive cell proliferation assay (Promega).
Immunoprecipitation and Immunoblotting--
Cells (2 × 107) were cultured overnight in the absence of growth
factor and left unstimulated. Cells were washed with cold PBS
containing 20 mM sodium orthovanadate and lysed on ice in lysis buffer (50 mM Hepes (pH 7.5), 150 mM
NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, 2 mg/ml iodoacetamide, 0.2 mg/ml trypsin inhibitor (Boehringer Mannheim), and
CompleteTM protease inhibitor (Boehringer Mannheim)) for 15 min. Insoluble material was removed by centrifugation, and cell lysates
were incubated with primary antibody for 2 h at 4 °C.
Antibodies used for immunoprecipitation were the anti-h
Immunoprecipitates were separated by SDS-polyacrylamide gel
electrophoresis on 10% gels and electrophoretically transferred to
PolyScreenR polyvinylidene difluoride membranes (NEN Life
Science Products). Membranes were then incubated with the anti-h Isolation of Factor-independent BAF/I374N Cells Infected with an
FDC-P1 cDNA Retroviral Expression Library--
We have previously
identified a constitutively activating point mutation, I374N, in the
extracellular region of h
Using procedures described previously (25), an FDC-P1 cDNA library
(~8.5 × 105 independent plasmid clones, with an
average insert size of 1.1 kb) was generated in the pRUFNeo retroviral
expression vector. As described under "Experimental Procedures,"
the plasmid DNA was used to generate a stable
BAF-B03 cells expressing I374N (BAF/I374N) were infected by
co-cultivation with the virus-producing PCR Recovery of Mouse GMR Expression of mGMR
The observation that the mouse GMR The I374N Mutation Induces Constitutive Association of h
The constitutive association of mGMR Both the N-terminal and C-terminal Regions of mGMR
Considering that the cytoplasmic domain of GMR
We next examined whether the inability of the extracellular and
cytoplasmic truncation mGMR Species Specificity of GMR The I374N Mutant Confers Factor Independence on Human Hemopoietic
Cells: A Possible Role for hGMR
Unfortunately, to the best of our knowledge, no human
factor-dependent hemopoietic cell lines "equivalent" to
BAF-B03 cells, i.e. that lack human GMR Constitutive Activation of I374N in Mouse Cells Requires
mGMR Physical Association of I374N and
mGMR
We observed that deletions in the extracellular N-terminal domain of
mGMR
Our observation that the cytoplasmic domain of GMR
Most importantly, however, the fact that (i) mGMR Determinants of the Species-specific Requirement for GMR
Our studies with mouse/human chimeric GMR
The relevance of the requirement for GMR
Finally, the prediction that the extracellular (I374N) and
transmembrane (V449E) constitutive mutants of h-subunit (hGMR
) and a common signal-transducing
-subunit (h
c) that is shared with the interleukin-3 and -5 receptors. We have previously identified a constitutively active
extracellular point mutant of h
c, I374N, that can confer factor
independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells
(Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287). This restricted activity
suggested the involvement of cell type-specific signaling molecules in
the activation of this mutant. We report here that one such molecule is
the mouse GMR
(mGMR
) subunit, since introduction of mGMR
, but
not hGMR
, into BAF-B03 or CTLL-2 cells expressing the I374N mutant
conferred factor independence. Experiments utilizing mouse/human
chimeric GMR
subunits indicated that the species specificity lies in
the extracellular domain of GMR
. Importantly, the requirement for
mGMR
correlated with the ability of I374N (but not wild-type h
c)
to constitutively associate with mGMR
. Expression of I374N in human
factor-dependent UT7 cells also led to factor-independent
proliferation, with concomitant up-regulation of hGMR
surface
expression. Taken together, these findings suggest a critical role for
association with GMR
in the constitutive activity of I374N.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit (hGMR
)
associated with a common signal-transducing
-subunit (h
c) that is
also utilized by the IL-3 and IL-5 receptors (2-6), all of which
belong to the cytokine receptor family (reviewed in Ref. 7). Members of
this family are characterized by a structurally conserved extracellular
cytokine receptor module (CRM) of about 200 amino acids that consists
of two fibronectin type III-like domains (8). The
-subunit has two
CRMs, whereas the
-subunits contain one CRM and an additional N-terminal domain of about 100 amino acids.
-
-subunit heterodimerization is a key step in the
formation of these complexes (9, 10). More recently, it has been shown
that
-subunit homodimers are found in active hGMR (11) and human
IL-3R (12) complexes and that the functional hGMR complex may contain
at least two
-subunits (13). Taken together, these results suggest
that the
- and
-subunits may form higher order receptor
complexes, and indeed it has been proposed that the GMR/IL-3R/IL-5R
normally functions as an
2
2 tetramer (10,
12, 13).
c by virtue of their ability to
confer factor-independent proliferation on mouse factor-dependent FDC-P1 cells (16, 17). One of these
mutations, V449E, is located in the transmembrane domain of h
c and
is similar to an activating mutation in the
neu/c-erbB-2 oncogene (18, 19). By analogy, this
mutant most likely acts by inducing h
c homodimerization. Another
group of activating point mutations, exemplified by I374N, lies in the
extracellular region of h
c; however, it is unclear precisely how
this group might affect receptor function. Interestingly, only certain
transmembrane mutants, such as V449E, were able to confer factor
independence on mouse factor-dependent BAF-B03 cells,
suggesting that the I374N mutation activates h
c in a cell
type-specific manner.
(mGMR
)
subunit as one such molecule and show that one effect of the I374N
mutation is to induce constitutive association with mGMR
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 (21) ecotropic retroviral
packaging cell lines were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. The BING amphotropic
retroviral packaging cell line was kindly provided by Prof. Suzanne
Cory (Walter and Eliza Hall Institute, Melbourne, Victoria, Australia) with permission from Dr. Warren Pear (MIT, Cambridge, MA) and was
maintained as described above. The CTL-EN subline of the mouse IL-2-dependent cell line, CTLL-2 (22), was kindly provided
by Dr. John Norton (Paterson Institute for Cancer Research, Manchester) and was maintained as described previously for CTLL-2 cells (16). Mouse
IL-3-dependent BAF-B03 cells (23) were maintained as
described previously (16). Human factor-dependent UT7 cells
(24) were maintained in Dulbecco's modified Eagle's medium plus 10%
fetal calf serum supplemented with 2 ng/ml human GM-CSF.
2 packaging cells. Infected
2 cells were
harvested and selected in medium containing G418 (400 µg/ml) to
generate the stable G418-resistant
2 retroviral library. BAF-B03
cells expressing the I374N h
c mutant were infected with the
2
retroviral library by co-cultivating 3.75 × 105
BAF/I374N cells with 1.2 × 106 irradiated (30 grays)
2 cells for 48 h in each of eight 25-cm2 culture
flasks. The BAF/I374N cells were then harvested, washed, and selected
for factor-independent growth in 24-well multidishes (204 wells, each
seeded with 105 cells) in liquid culture medium without factor.
expression construct was generated by subcloning the full-length
mGMR
cDNA recovered from factor-independent BAF/I374N infectants
into the BamHI and HindIII restriction sites of
pRUFNeo. The pRUFNeo/hGMR
expression construct was generated by
inserting the cDNA for hGMR
into the XhoI site of pRUFNeo.
(FmGMR
), a 5'
BamHI/NaeI fragment encoding the signal sequence and first 8 structural residues of mGMR
was excised from
pRUFNeo/mGMR
and replaced in frame with a PCR-generated
BamHI/NaeI fragment from
pcDNA1Neo/FhIL-3R
(kindly provided by Richard
D'Andrea, Hanson Center for Cancer Research, Adelaide, South
Australia, Australia) encoding the hIL-3R
signal sequence, FLAG
octapeptide, and first 6 structural residues of hIL-3R
. The sense
primer corresponded to the T7 promoter sequence and included a
BamHI site, and the antisense primer corresponded to codons
19-24 (as numbered by Kitamura et al. (5)) of hIL-3R
and
included a NaeI site. The pRUFPuro/FmGMR
expression vector was constructed by inserting the
BamHI/EcoRI FmGMR
cDNA from
pRUFNeo/FmGMR
into the BamHI and
EcoRI sites of the pRUFPuro retroviral expression vector
(16).
-subunits (between residues Cys16 and Trp17
as numbered by Hayashida et al. (4)) by site-directed
mutagenesis using the pAlter-1 system (Promega) in accordance with the
manufacturer's instructions. The modified
-subunit cDNAs were
subcloned into the BamHI and HindIII restriction
sites of pRUFNeo.
chimeras were generated by PCR amplification and
ligation of the relevant portions of human and mouse GMR
: (i) the
pRUFNeo/h
m
1 chimera encoding the extracellular and transmembrane domains of hGMR
(346 amino acids) and the cytoplasmic domain of
mGMR
(38 amino acids); (ii) the pRUFNeo/h
m
2 chimera encoding the extracellular N-terminal domain of hGMR
(117 amino acids) and
the extracellular CRM, transmembrane, and cytoplasmic domains of
mGMR
(262 amino acids); (iii) the pRUFNeo/Fm
h
1
chimera encoding the extracellular and transmembrane domains of
FmGMR
(335 amino acids) and the cytoplasmic domain of
hGMR
(54 amino acids); and (iv) the pRUFNeo/Fm
h
2
chimera encoding the extracellular FLAG-tagged N-terminal domain of
FmGMR
(111 amino acids) and the extracellular CRM,
transmembrane, and cytoplasmic domains of hGMR
(283 amino acids). A
full description of the templates and primers used is available upon request.
were generated by PCR on the
pRUFNeo/FmGMR
construct with primers designed to amplify
the entire construct except for the desired extracellular sequence to
be removed while leaving the N-terminal signal sequence and FLAG
octapeptide intact. Each PCR was performed with different sense primers
corresponding to codons 97-102 (for m
D1) and codons 195-200 (for
m
D2) and the same antisense primer corresponding to codons 9-14 of
mGMR
. The blunt ends of each PCR fragment were then ligated together in frame.
was generated by PCR on
the pRUFNeo/FmGMR
construct with RCF1 as the sense
primer and an antisense primer that contained codons 344-339 of the
mGMR
cytoplasmic domain together with a HindIII
restriction site and termination codon. The PCR products were subcloned
into the BamHI and HindIII restriction sites of pRUFNeo.
2 packaging cells (16) or transiently transfected BOSC
23 packaging cells as described previously (27). Infected BAF-B03 cells
were selected in liquid culture medium containing growth factor and
either G418 (1.5 mg/ml) or puromycin (2 µg/ml). Infected CTL-EN cells
were selected as described previously for CTLL-2 cells (16).
subunits was detected by
staining with the anti-FLAG monoclonal antibody M2 (Kodak), and
expression of hGMR
subunits was detected by staining with the
anti-hGMR
monoclonal antibody 8G6 (29). Expression of wild-type and
I374N mutant
-subunits on the surface of infected BAF-B03 cells was
detected by staining with the anti-h
c monoclonal antibody 1C1 (10),
whereas HSV epitope-tagged wild-type and I374N mutant
-subunits
expressed on the surface of human UT7 cells were detected by staining
with an HSV tag monoclonal antibody (Novagen).
c antibody
8E4 (30) and the anti-FLAG antibody M2 (Kodak). Immune complexes were
precipitated with 75 µl of protein A-Sepharose (Amersham Pharmacia
Biotech) for 1 h at 4 °C, washed three times with lysis buffer,
and boiled in 1× reducing SDS sample buffer. In the case of whole cell
protein analyses, samples were lysed in buffer without 10% glycerol,
and insoluble material was removed and boiled in 1× reducing SDS
sample buffer.
c
antibody 1C1 (10), the anti-hGMR
antibody 8D10 (29), or the
biotinylated anti-FLAG antibody BIOM2 (Kodak), as indicated, following
which the membranes were washed and incubated with either an alkaline
phosphatase-conjugated anti-mouse antibody (Amersham Pharmacia Biotech)
or a streptavidin-conjugated alkaline phosphatase antibody (Molecular
Probes, Inc., Eugene, OR), as appropriate. Membranes were washed and
subjected to enhanced chemifluorescence detection (Amersham Pharmacia
Biotech) as per the manufacturer's instructions, following which they
were scanned on a FluorImager (Molecular Dynamics, Inc., Sunnyvale,
CA). For reprobing, membranes were stripped in 50 mM Tris
(pH 7.4), 2% SDS, 100 mM
-mercaptoethanol at 55 °C
for 20 min; washed; and subsequently probed with the indicated antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
c by virtue of its ability to confer
factor-independent growth on FDC-P1 cells (16). Surprisingly, this
mutant was unable to confer factor independence on mouse
IL-3-dependent BAF-B03 cells, leading us to suggest that
the cell type-specific activity of this mutant may reflect the presence
of a
-subunit-associated signaling molecule in FDC-P1 cells, but not
in BAF-B03 cells, that is required by this mutant for constitutive
activation (16). We therefore reasoned that the introduction of such a
molecule from FDC-P1 cells into BAF-B03 cells expressing the I374N
mutant should lead to its constitutive activity and thus render these
cells factor-independent.
2 retroviral library
estimated to contain ~3.5 × 106 independent viral
producer clones, which should adequately represent all cDNA species
present in the plasmid library.
2 cells at an infection frequency of 18% (estimated by colony assays in the presence of G418).
As a control, parallel infections were also performed on uninfected
BAF-B03 cells and BAF-B03 cells expressing wild-type h
c. Cells were
then selected for factor-independent growth in 24-well multidishes.
After 1 week in the absence of factor, 37 of 204 wells seeded with
105 infected BAF/I374N cells contained viable,
proliferating cells, while no such cells were present in control
cultures. Factor independence was not the result of autocrine growth
factor production, since conditioned medium from the factor-independent
cell cultures did not support the growth of uninfected BAF-B03 cells
(data not shown).
cDNA from Factor-independent
BAF/I374N Infectants--
To identify the cDNA sequence carried by
the provirus in the factor-independent BAF/I374N infectants, long range
PCR was performed with retroviral primers on genomic DNA samples from
17 of the 37 factor-independent cell populations. This revealed a
common fragment of approximately 2.3 kb that was amplified from all 17 genomic DNA samples (data not shown); considering the positions of the
PCR primers relative to the cloning sites in pRUFNeo, the size of the
cDNA insert was estimated to be 1.9 kb. For 8 of the 17 samples,
the 2.3-kb fragment was the only PCR product generated, suggesting that
these factor-independent cell populations contained only one retroviral
insertion and that its presence was responsible for factor
independence. Sequence analysis of the 1.9-kb cDNA insert recovered
from two of the factor-independent cell populations revealed that it
corresponded to the full-length cDNA for the mGMR
subunit
(31).
with I374N in BAF-B03 and CTL-EN Cells
Results in Factor Independence--
To confirm that mGMR
would
allow the constitutive activation of I374N, we expressed the recovered
mGMR
subunit in BAF/I374N cells and then tested these cells for
factor independence. In order to monitor cell surface expression of
mGMR
, a FLAG epitope-tagged mGMR
(FmGMR
) was
generated in the pRUFNeo vector (see "Experimental Procedures").
This was introduced into puromycin-resistant BAF/I374N cells as well as
wild-type h
c-expressing and uninfected BAF-B03 cells. Following
selection for G418 resistance, flow cytometric analysis with a
FLAG-specific monoclonal antibody indicated that the
FmGMR
subunit was efficiently expressed on the surface
of these cells (Fig. 1A). Upon
selection for growth in medium without factor, only BAF-B03 cells
co-expressing FmGMR
and I374N exhibited
factor-independent growth (Fig. 1B). The ability of
FmGMR
to behave as wild-type mGMR
was demonstrated by
the proliferation of all FmGMR
-infected BAF-B03 cells in
response to mGM-CSF (Fig. 1B).
View larger version (31K):
[in a new window]
Fig. 1.
Co-expression of
FmGMR with I374N confers factor
independence on BAF-B03 cells. A, flow cytometric
analysis of FmGMR
and
-subunit expression on
G418-selected BAF-B03 cells. Uninfected BAF-B03 cells
(uninf) or cells expressing either wild-type (wt)
or I374N
-subunits were infected with a retrovirus encoding the
FmGMR
subunit and stained with an irrelevant control
antibody (dashed line), the anti-h
c antibody
1C1 (thin solid line), and the
anti-FLAG antibody M2 (thick solid
line) by high sensitivity immunofluorescence. Cell number
and fluorescence are in arbitrary units; the latter is plotted on a
logarithmic scale. Also shown are analyses of cells not exposed to the
FmGMR
virus. B, proliferation of the BAF-B03
cells depicted in A in the presence of mIL-3 (300 units/ml)
or mGM-CSF (80 units/ml) or in the absence of either factor, as
indicated. Proliferation assays were carried out, as described under
"Experimental Procedures," with 5 × 103 cells
plated in triplicate. Error bars indicate the S.E. of the
mean of each triplicate.
subunit was required for the
activity of I374N raised the possibility that another component(s) of
the mouse GMR or IL-3R (i.e. mIL-3R
m
c or m
IL-3)
present in FDC-P1 and BAF-B03 cells might also be needed. We therefore introduced I374N and, as a control, wild-type h
c with
FmGMR
into mouse IL-2-dependent CTL-EN
cells, which do not express any receptor components belonging to the
GMR or IL-3R. CTL-EN cells are a derivative of CTLL-2 cells engineered
for increased expression of the ecotropic retroviral receptor
(41),2 thereby rendering them
more susceptible to retroviral infection. We also included the V449E
transmembrane h
c mutant in this experiment, since it is inactive
when expressed in CTLL-2 cells, although, unlike the I374N mutant, it
does confer factor independence on BAF-B03 cells (16). The expression
of these subunits was confirmed by flow cytometry (data not shown),
following which these cells were tested for factor-independent
proliferation. As shown in Fig. 2, only
CTL-EN cells expressing both FmGMR
and I374N were
factor-independent, thereby indicating that components of the mouse
IL-3R are not required for the constitutive activity of I374N. In view
of this result, all subsequent experiments were performed in BAF-B03
cells.
View larger version (12K):
[in a new window]
Fig. 2.
Factor-independent proliferation of CTL-EN
cells co-expressing FmGMR and the
I374N mutant. Proliferation of CTL-EN cells expressing the
indicated subunits in the absence of factor. Cells were maintained in
mouse IL-2 (4 ng/ml) and the appropriate drug selection prior to assay
for factor-independent growth.
c with
mGMR
in BAF-B03 Cells--
To examine whether the requirement for
mGMR
by I374N might reflect a physical association between these two
subunits, BAF-B03 cells co-expressing FmGMR
with I374N
or, as a control, wild-type h
c were subjected to immunoprecipitation
with an anti-h
c antibody, followed by immunoblot analysis with an
anti-FLAG antibody. As shown in Fig. 3A, a protein of 60-kDa,
consistent with the predicted size of mGMR
, was detected only in
immunoprecipitates from cell lysates expressing FmGMR
and the I374N mutant. Importantly, the converse immunoprecipitation (with anti-FLAG antibody) and immunoblot analysis (with anti-h
c antibody) confirmed the physical association between mGMR
and the
I374N mutant (data not shown). Reprobing the immunoblot with an
anti-h
c antibody indicated that both wild-type and I374N
-subunits were immunoprecipitated from the appropriate cell lysates
(Fig. 3B). Furthermore, immunoblot analysis of whole cell
lysates with an anti-FLAG antibody indicated that the total levels of
FmGMR
protein present in lysates from all cell
populations were comparable (Fig. 3C). Together, these
observations indicate that the I374N mutation acts, at least in part,
by inducing constitutive association of h
c with mGMR
.
View larger version (39K):
[in a new window]
Fig. 3.
Co-immunoprecipitation of
FmGMR and I374N from BAF-B03
cells. A and B, uninfected BAF-B03 cells and
cells expressing the indicated subunits were incubated in medium
without factor, and lysates were immunoprecipitated (IP)
with the anti-h
c antibody 8E4. Immunoprecipitated proteins were
analyzed by immunoblotting (IB) with the anti-FLAG antibody
M2 (A) or with the anti-h
c antibody 1C1 (B).
C, whole cell lysates from the indicated BAF-B03 cells were
subjected to immunoblotting with the anti-FLAG antibody M2.
with the I374N mutant was
reminiscent of the ability of human GMR
to associate with wild-type
h
c in the absence of GM-CSF (32). We therefore examined the ability
of I374N to associate with hGMR
in the absence of ligand, since a
failure to do so could explain our previous observation that
co-expression of hGMR
did not allow constitutive activity of I374N
in BAF-B03 cells (Refs. 16 and 27; Fig. 7). The experiment illustrated
in Fig. 4A shows, however,
that both mutant and wild-type h
c could associate equally well with
hGMR
in the absence (or presence) of ligand, as judged by
co-immunoprecipitation from BAF-B03 cells expressing both subunits.
Equivalent levels of expression of the
- and
-subunits are
confirmed by the analyses of Fig. 4, B and C,
respectively.
View larger version (40K):
[in a new window]
Fig. 4.
Ligand-independent association of
hGMR with I374N and wild-type
h
c. A and B,
uninfected BAF-B03 cells and cells expressing the indicated hGMR
subunits were incubated in medium with (+) or without (
) hGM-CSF (10 ng/ml), and lysates were immunoprecipitated (IP) with the
anti-h
c antibody 8E4. Immunoprecipitated proteins were analyzed by
immunoblotting (IB) with the anti-hGMR
antibody 8D10
(A) or with the anti-h
c antibody 1C1 (B).
C, whole cell lysates from the indicated BAF-B03 cells were
subjected to immunoblotting with the anti-hGMR
antibody 8D10.
Are Essential
for Activation of and Association with the I374N Mutant--
To
broadly define the regions of the mGMR
extracellular domain required
for the constitutive activation of I374N, two FLAG-tagged extracellular
truncation mutants were generated. One of these, Fm
D1,
lacked residues Leu15-Ala96, which comprise the
N-terminal domain, whereas the other, Fm
D2, lacked
residues Leu15-Glu194, which also includes
domain 1 of the cytokine receptor module (CRM; Fig. 4A).
Although these truncation mutants (and full-length FmGMR
) were efficiently expressed on the surface of
G418-resistant cells (Fig. 4B), neither truncation mutant
was able to confer factor independence on BAF/I374N cells (Fig.
4C), indicating that the N-terminal domain of mGMR
is
required for constitutive activation of I374N. Furthermore, the
inability of BAF/I374N cells expressing the m
D1 mutant to
proliferate in the presence of mGM-CSF suggests that the N-terminal
domain of mGMR
is also important in normal mGMR function.
is essential for
normal GM-CSF-mediated cell growth (33), we also investigated whether
the cytoplasmic domain of mGMR
was required for constitutive signaling by I374N. We therefore generated a cytoplasmic truncation mutant, Fm
t3, which lacked the C-terminal 14 amino acids
of mGMR
(Fig. 5A). Although
G418-resistant BAF/I374N infectants efficiently expressed
Fm
t3 (Fig. 5B), these cells failed to grow in
the absence of factor (Fig. 5C) or in response to mGM-CSF.
This implies that the C-terminal 14 amino acids of mGMR
are
essential for mediating factor-independent growth conferred by I374N
and also for normal mGM-CSF-mediated growth.
View larger version (32K):
[in a new window]
Fig. 5.
Analysis of BAF/I374N cells expressing
FmGMR extracellular and
cytoplasmic truncation mutants. A, schematic
illustration of the truncated FmGMR
subunits showing the
regions deleted in each truncation mutant. For comparison, the
full-length FmGMR
is also shown. The
asterisks represent the C terminus of the depicted subunits.
B, flow cytometric analysis of BAF/I374N cells superinfected
with full-length and truncated FmGMR
subunits.
Procedures, nomenclature, and axes are as in Fig. 1A. Also
shown are "parental" BAF/I374N cells. C, proliferation
assay of the BAF/I374N cells depicted in B in the presence
of mIL-3 (300 units/ml) or mGM-CSF (80 units/ml) or in the absence of
either factor, as indicated.
mutants to confer factor independence on
BAF/I374N cells was due to a failure to associate with I374N. Lysates
from BAF/I374N cells expressing the Fm
D1 extracellular
truncation and the Fm
t3 cytoplasmic truncation were
therefore subjected to immunoprecipitation with an anti-FLAG antibody,
followed by immunoblot analysis with an anti-h
c antibody. As shown
in Fig. 6A, the I374N mutant
was precipitated when co-expressed with the full-length
FmGMR
subunit but not with the truncated
FmGMR
subunits. Reprobing with an anti-FLAG antibody
demonstrated that both full-length and truncated FmGMR
subunits were themselves immunoprecipitated (Fig. 6B), and immunoblot analysis of whole cell lysates with an anti-h
c antibody confirmed that comparable levels of the I374N mutant were expressed in
the cells (Fig. 6C). Thus, these data demonstrate that both the N-terminal and C-terminal regions of mGMR
are essential for the
association with I374N and, together with the data presented in Fig. 5,
that the constitutive activity of I374N is dependent upon this
association.
View larger version (33K):
[in a new window]
Fig. 6.
Extracellular and cytoplasmic truncations of
FmGMR abolish the
co-immunoprecipitation of FmGMR
and I374N from BAF-B03 cells. A and B,
the BAF-B03 cells expressing the indicated subunits were incubated in
medium without factor and lysates were immunoprecipitated
(IP) with the anti-FLAG antibody M2. Immunoprecipitated
proteins were analyzed by immunoblotting (IB) with the
anti-h
c antibody 1C1 (A) or with the anti-FLAG antibody
M2 (B). The asterisk by Fm
in
B indicates that the small C-terminal deletion mutant
Fm
t3 ran at the same size as the full-length
FmGMR
under the gel conditions employed. C,
whole cell lysates from the indicated BAF-B03 cells were subjected to
immunoblotting with the anti-h
c antibody 1C1.
for the Constitutive Activation of
I374N Lies in Its Extracellular and/or Transmembrane Domains--
In
view of our previous observations that co-expression of the human
GMR
subunit with I374N in BAF-B03 and CTLL-2 cells did not lead to
factor-independent growth (16, 27), the ability of the mouse GMR
subunit to facilitate constitutive activity of I374N in BAF-B03 and
CTL-EN cells was somewhat surprising. To define which region(s) of the
GMR
subunit govern this apparent species specificity, we constructed
a series of chimeric GMR
subunits containing regions from both
species (Fig. 7A). These chimeras, along with the normal FmGMR
and hGMR
subunits, were then introduced into BAF/I374N cells and tested for
their ability to confer factor independence. Flow cytometric analyses
confirmed that while the chimeric GMR
subunits were co-expressed
with the I374N mutant (Fig. 7B), only cells co-expressing
the Fm
h
1 chimera or, as expected, the normal
FmGMR
subunit with the I374N mutant exhibited
factor-independent proliferation (Fig. 7C). Thus, the
species specificity lies in the extracellular and/or transmembrane
domains of mGMR
. Furthermore, since chimeras containing only the
mouse N-terminal domain (Fm
h
2) or the mouse
extracellular CRM and transmembrane domain (h
m
2) were unable to
confer factor independence on BAF-B03 cells, it is likely that both of
the mGMR
regions present in these chimeras contribute to the
species-specific requirement for mGMR
for I374N activity.
View larger version (37K):
[in a new window]
Fig. 7.
Analysis of BAF/I374N cells infected with
retroviruses encoding chimeric mouse and human GMR
subunits. A, schematic illustration of chimeric
GMR
subunits. Regions from the mouse GMR
are shown in
white, whereas regions from the human GMR
are shown in
black. For comparison, the normal FmGMR
and
hGMR
subunits are also shown. B, flow cytometric analysis
of BAF/I374N cells that were superinfected with retroviruses encoding
normal and chimeric GMR
subunits and stained with the anti-FLAG
antibody M2 (dotted line), the anti-hGMR
antibody 8G6 (thick solid line), and
the anti-h
c antibody 1C1 (thin solid
line). Axes are as in Fig. 1A.
C, proliferation assay of the BAF/I374N cells depicted in
B in the presence of mIL-3 (300 units/ml), mGM-CSF (80 units/ml), or hGM-CSF (1 ng/ml) or in the absence of any factor, as
indicated.
in the Constitutive Activity of
I374N in Human Cells--
Although the human GMR
subunit was unable
to facilitate the constitutive activity of I374N in mouse BAF-B03 and
CTLL-2 cells (16, 27) (see also Fig. 7), it was conceivable that the
I374N mutant might be constitutively active in human cells expressing hGMR
. We therefore introduced this mutant and, as a control, wild-type h
c into human
GM-CSF/IL-3/erythropoietin-dependent UT7 cells and tested
these cells for factor-independent proliferation. To distinguish
between the introduced
-subunits and the endogenous
-subunits
expressed by UT7 cells, we inserted an 11-amino acid HSV-derived
epitope at the N terminus of both wild-type and I374N
-subunits.
Cells infected with these modified
-subunits were then selected for
G418 resistance or growth in medium without factor. The surface
expression of the introduced subunits was confirmed by flow cytometric
analysis of infected cells stained with both anti-h
c and anti-HSV
antibodies (Fig. 8A). In two
independent experiments, one of which is shown in Fig. 8B,
the I374N mutant allowed factor-independent proliferation of UT7 cells.
Factor independence was not the result of low level autocrine growth factor production, since conditioned medium from factor-independent cell pools did not support the growth of uninfected UT7 cells (data not
shown).
View larger version (23K):
[in a new window]
Fig. 8.
Analysis of human UT7 cells infected with the
I374N mutant. A, flow cytometric analysis of G418-resistant UT7
cells infected with retroviruses encoding the HSV-tagged (as shown by
asterisks) wild-type and I374N -subunits, except for the
panel labeled FI *I374N, which shows staining of
cells infected with the HSV-tagged I374N mutant and selected for
factor-independent growth. Also shown are analyses of uninfected UT7
cells. Cells were stained with an irrelevant control antibody
(dashed line), the anti-h
c antibody 1C1
(thin solid line), and the anti-HSV
antibody (dotted line) by high sensitivity
immunofluorescence. The axes are as in Fig. 1A.
B, proliferation assay of the UT7 cells depicted in
A in the presence and absence of human GM-CSF (2 ng/ml).
C, flow cytometric analysis of hGMR
expression on the
surface of the UT7 cells depicted in A. Cells were stained
with the anti-hGMR
antibody 8G6 by high sensitivity
immunofluorescence. The axes are as in Fig.
1A.
, are available;
thus, we could not directly test the requirement for human GMR
by
I374N in human hemopoietic cells. Notably, however, flow cytometric
analysis with an anti-hGMR
antibody revealed that the expression of
hGMR
was significantly up-regulated on the surface of
factor-independent cells expressing I374N (FI *I374N cells)
compared with uninfected cells or G418-resistant cells (expressing
wild-type h
c or I374N) that were not selected for factor
independence (Fig. 8C). Importantly, the increase in hGMR
expression specifically correlated with the factor independence of
I374N-expressing cells. This increase in hGMR
expression was not
simply a function of high level
-subunit expression (see FI
*I374N histogram in Fig. 8A), since infected UT7 cells
that were sorted for comparably high levels of HSV-tagged wild-type h
c exhibited a similar low level of hGMR
expression to the
unsorted cells (*wt) shown in Fig. 8C (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The I374N mutation in the extracellular domain of h
c
confers factor independence on mouse FDC-P1 cells but not BAF-B03 or CTLL-2 cells (16), raising the possibility that cell type-specific signaling molecules are involved in its activation. In this study, we
have employed retroviral expression cloning to identify the mGMR
subunit as one such molecule, since its introduction into BAF-B03 and
CTL-EN (a derivative of CTLL-2) cells expressing the I374N mutant
conferred factor independence. Importantly, the absence of the mouse
GMR and IL-3R in CTL-EN cells indicates that the mechanism of
activation of I374N does not require any subunits, apart from mGMR
,
of these receptors. In contrast, another h
c mutant, V449E, that
confers factor independence on both FDC-P1 and BAF-B03 cells (16) is
not constitutively active when co-expressed with mGMR
in CTL-EN
cells. This suggests that the I374N and V449E mutants are activated by
fundamentally different mechanisms.
--
Co-immunoprecipitation experiments demonstrated that one
effect of the I374N mutation in h
c is to induce constitutive
association with mGMR
. The constitutive association between these
subunits is reminiscent of a recent report in which hGMR
and
wild-type h
c were co-immunoprecipitated from cell lines in the
absence of GM-CSF (32). Factor-independent association with h
c
appears to be a unique property of GMR
, since similar preformed
complexes could not be detected with hIL-3R
or hIL-5R
(32). This
may in part explain the specific requirement for mGMR
, as opposed to
mIL-3R
, for constitutive activity of I374N.
abolished both the constitutive activity of I374N and the
association between I374N and mGMR
, as well as mGM-CSF-induced proliferative signaling. While the corresponding domains of the hIL-3R
and hIL-5R
subunits have been reported to play a critical role in ligand binding (34-36), our demonstration that the N-terminal domain of mGMR
is required for association with the h
c mutant suggests that this domain may also play a role in receptor subunit assembly.
is needed for the
activity of I374N was not unexpected, since deletion of the cytoplasmic
domains of GMR
, IL-3R
, and IL-5R
renders these receptors
inactive in proliferative signaling (33, 34, 37). Normally, however,
-subunit cytoplasmic truncations do not detectably affect the
association of
- and
-subunits, since truncated
-subunits
still form high affinity ligand-binding receptors (33, 34, 37), and a
cytoplasmic truncation of hGMR
could still associate with h
c in
the preformed hGMR complex described by Woodcock et al.
(32). Thus, it is surprising that deletion of the C-terminal 14 amino
acids of mGMR
also abolished the association between mGMR
and
I374N. Nevertheless, this observation suggests that there may be a
degree of interaction between the intracellular domains of
- and
-subunits and that the effect of such an interaction may only be
detectable in the context of weaker extracellular interactions between
mGMR
and I374N as compared with those between wild-type h
c and
hGMR
.
associates with
the I374N mutant but not with wild-type h
c and (ii) association of
mGMR
mutants with I374N correlates with their ability to allow constitutive receptor activity suggests that induction of this association is essential for h
c activation. However, constitutive association of hGMR
with h
c per se is not sufficient
for receptor activation (32); thus, it is likely that the I374N
mutation has additional effects such as mimicking a ligand-induced
conformational change in h
c, as we have suggested previously (27,
38).
for the Constitutive Activity of I374N--
In view of the ability of
mouse GMR
to allow constitutive activity of I374N in mouse cells, it
is somewhat surprising that co-expression of the human GMR
subunit
with I374N in mouse BAF-B03 and CTLL-2 cells does not lead to
factor-independent proliferation (Refs. 16 and 27; see also Fig.
7C). This is not due to the inability of I374N to interact
with hGMR
because their co-expression in BAF-B03 and CTLL-2 cells
results in the formation of a high affinity receptor and generation of
a proliferative signal in response to human GM-CSF (16, 27). Moreover,
I374N, like wild-type h
c (32), also efficiently
co-immunoprecipitates with hGMR
in the absence of hGM-CSF (Fig.
4).
subunits showed that only
the chimera containing the entire extracellular and transmembrane domains of mGMR
conferred factor independence on BAF/I374N cells, while in contrast, the human and mouse cytoplasmic domains were interchangeable. This suggests two possible explanations for species specificity: (i) that the extracellular domain of mGMR
interacts with I374N in a different manner from that of its human homologue, to
allow formation of an active complex in the absence of ligand or (ii)
that mGMR
interacts with a membrane-spanning accessory signaling
molecule in a species-specific manner. The latter explanation would
also suggest that species specificity might also be a function of the
host cell species, i.e. that the accessory molecule might interact preferentially with the GMR
subunit of the same species. However, the fact that the I374N mutant was able to confer
factor-independent proliferation on human
GM-CSF/IL-3/erythropoietin-dependent UT7 cells argues
against an exclusive requirement for murine GMR
. Moreover, the
expression of hGMR
on the surface of the factor-independent cells
was significantly up-regulated, suggesting that selection for factor
independence also selected for hGMR
subunit expression. This is
consistent with the notion that GMR
is involved in the constitutive
activation of I374N in these cells also and that the species-specific
requirement of GMR
for the constitutive activity of I374N may
reflect the species of cell in which the mutant is expressed.
by I374N, and indeed other
extracellular h
c mutants, to the activity of these mutants in
primary hemopoietic cells should be noted. Retroviral infection of
mouse fetal liver progenitors with the I374N mutant and another extracellular h
c mutant (FI
; Ref. 39) results in the formation of
factor-independent cells of only granulocytic and monocytic lineages
(40), the two major lineages controlled by GM-CSF (reviewed in Ref. 1).
This observation is consistent with the notion that the constitutive
activity of I374N and other extracellular h
c mutants is restricted
to cells expressing GMR
.
c appear to act (at
least in part) by inducing
-
or
-
dimerization,
respectively, is interesting in light of evidence that the wild-type
GMR/IL3R/IL5R may contain both
-
and
-
dimers (see the
Introduction). It is tempting to speculate that each class of mutant
might activate a subset of the multiple, overlapping signaling pathways
activated by the wild-type receptor complex. Thus, they may constitute
useful tools for dissecting receptor signaling.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. John Rayner for assistance with construction of the FDC-P1 cDNA retroviral expression library, Sun Qiyu and Prof. Angel Lopez for supplying anti-receptor antibodies, and various colleagues who generously supplied growth factors. We thank Alan Bishop and Sandy McIntyre for assistance with flow cytometry and Arthur Mangos for automated sequencing analyses. We also thank Dr. John Norton (Paterson Institute for Cancer Research, Manchester) for the CTL-EN cell line and Dr. Richard D'Andrea for discussions and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a research grant from the National Health and Medical Research Council (NHMRC) of Australia.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.
Recipient of a Dawes postgraduate scholarship from the Royal
Adelaide Hospital. Present address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109-1024.
§ A Senior Research Fellow of the NHMRC. To whom correspondence should be addressed: Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Rd., Adelaide, South Australia 5000, Australia. Tel.: 61-8-8222-3305; Fax: 61-8-8232-4092; E-mail: Tom.Gonda{at}imvs.sa.gov.au.
2 J. Norton, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GM-CSF
granulocyte-macrophage colony-stimulating factor, mGM-CSF, mouse
GM-CSF;
GMR, GM-CSF receptor;
hGMR, human GMR;
mGMR, mouse GMR;
GMR, GMR
-subunit;
IL, interleukin;
hIL, human IL;
IL-3R and IL-5R, interleukin-3 and -5 receptors, respectively;
c, common
-subunit
of the GM-CSF, IL-3 and IL-5 receptors;
h
c, human
c;
CRM, cytokine receptor module;
PCR, polymerase chain reaction;
kb, kilobase pair(s);
wt, wild type;
HSV, herpes simplex virus.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|