From the Cytokine Receptor Laboratory, The Hanson Centre for Cancer Research, The Institute of Medical and Veterinary Science, Adelaide 5000, South Australia, Australia
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ABSTRACT |
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The human interleukin 3 (IL-3) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors
undergo covalent dimerization of the respective specific chains
with the common
subunit (
c) in the presence of
the cognate ligand. We have now performed alanine substitutions of
individual Cys residues in
c to identify the Cys
residues involved and their contribution to activation of the IL-3,
GM-CSF, and IL-5 receptors. We found that substitution of Cys-86,
Cys-91, and Cys-96 in
c but not of Cys-100 or Cys-234 abrogated disulfide-linked IL-3 receptor dimerization. However, although Cys-86 and Cys-91
c mutants retained their
ability to form non-disulfide-linked dimers with IL-3R
, substitution
of Cys-96 eliminated this interaction. Binding studies demonstrated that all
c mutants with the exception of C96A supported
high affinity binding of IL-3 and GM-CSF. In receptor activation
experiments, we found that
c mutants C86A, C91A, and
C96A but not C100A or C234A abolished phosphorylation of
c in response to IL-3, GM-CSF, or IL-5. These data show
that although Cys-96 is important for the structural integrity of
c, Cys-86 and Cys-91 participate in disulfide-linked
receptor heterodimerization and that this linkage is essential for
tyrosine phosphorylation of
c. Sequence alignment of
c with other cytokine receptor signaling subunits in
light of these data shows that Cys-86 and Cys-91 represent a motif
restricted to human and mouse
chains, suggesting a unique mechanism
of activation utilized by the IL-3, GM-CSF, and IL-5 receptors.
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INTRODUCTION |
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Cytokine receptor dimerization is a common theme in receptor activation (1). Following the binding of the cognate ligand to cytokine receptors, a sequential process takes place whereby receptor subunits associate and recruit cytoplasmic signaling molecules leading to receptor activation and cellular signaling (2). The general process of receptor dimerization exhibits variations among the cytokine receptor superfamily and may involve homodimerization or heterodimerization events depending on receptor subunit composition (3, 4). In the case of the growth hormone receptor, growth hormone binds initially to one receptor subunit and induces its homodimerization with a second, identical subunit (5). A similar process probably takes place with erythropoietin and granulocyte colony-stimulating factor, leading, in both cases, to receptor homodimerization and activation (6, 7).
With cytokine receptors that comprise multiple subunits, receptor
activation is accompanied by homodimerization or heterodimerization of
the signaling subunits. For example, in the
IL-61 receptor system, IL-6
induces dimerization of IL-6R with gp130 (8), homodimerization of
gp130, and receptor activation (9). On the other hand, the binding of
CNTF to CNTFR
induces its association with gp130 and the LIF
receptor, and the heterodimerization of gp130 and the LIF receptor is
accompanied by receptor activation (10). Similarly, heterodimerization
of IL-2R
and IL-2R
subunits is necessary for IL-2 receptor
activation (11, 12). Interestingly, in these cases, each receptor
chain constitutes the major binding subunit but does not seem to form
part of the signaling receptor complex.
The mechanism of activation of the GM-CSF/IL-3/IL-5 receptor system
exhibits features similar to the mechanism employed by the above
receptors, although some unique features are becoming evident. One of
the most important differences is the contribution that each receptor
chain makes to signaling. This is manifested in two ways: first,
unlike IL-6R
, CNTFR
, and the IL-2R
, the cytoplasmic domains of
GM-CSFR
, IL-3R
, and IL-5R
are all required for full receptor
activation and signaling (13-16). Second, IL-3R
and GM-CSFR
form
disulfide-linked dimers with the common
chain (
c) of
their receptor (4, 17). The disulfide-mediated dimerization of IL-3R
with
c and of GM-CSFR
with
c is
accompanied by tyrosine phosphorylation of
c (4). In all
of these cases, however, tyrosine phosphorylation is observed in the
disulfide-linked dimers as well as in the monomeric molecules, and
hence it is not clear which is the critical species for receptor
activation. Furthermore, the location of the cysteines involved in
disulfide linkage is not known, nor is it apparent whether they
constitute a functionally conserved motif in the cytokine receptor
superfamily. We have now performed single alanine substitutions of
candidate cysteine residues in the N-terminal cytokine receptor module
(CRM) of the IL-3, GM-CSF, and IL-5 receptor
c and
examined their contribution to disulfide-linked receptor dimerization,
high affinity ligand binding, and receptor activation.
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MATERIALS AND METHODS |
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Mutagenesis of Human c and Expression Plasmid
Constructs--
Cysteine residues were substituted with alanines in
the human
chain cDNA using oligonucleotide-directed mutagenesis
(Altered-sites, Promega, Sydney, New South Wales, Australia) as
described previously (18). The mutations were confirmed by nucleotide
sequencing, and the mutant
c cDNAs were subcloned
into the eukaryotic expression vector pcDNA1
(Invitrogen, San Diego, CA). The IL-3R, GM-CSFR, and IL-5R
chain
cDNAs were cloned into the eukaryotic expression vector
pCDM8 (Invitrogen) for transfection (18).
Cell Culture and DNA Transfection--
COS cells were maintained
in RPMI 1640 medium supplemented with 10% v/v fetal calf serum and
transfected by electroporation. Routinely, 2 × 107
COS cells were co-transfected in 0.8 ml of PBS at 0 °C with 25 µg
of wild type or mutated c cDNA together with 10 µg
of GM-CSFR and 10 µg of IL-3R
chain cDNA at 500 microfarads
with 300 V. After electroporation, cells were centrifuged through a
1-ml cushion of fetal calf serum, and cells were plated in either 25 ml
of medium/150-cm2 flask or 24-well plates for binding
analysis. Transfectants were incubated for 2 days prior to cytokine
treatment (18).
GM-CSF, IL-3, and IL-5-- Recombinant human IL-3, GM-CSF, and IL-5 were produced in Escherichia coli essentially as described before (21, 22). Cytokine purity and quantitation was determined by high performance liquid chromatography analysis. The unit activity of the cytokines based on the ED50 values in a proliferation assay (23) was 0.03 ng/ml GM-CSF, 0.1 ng/ml IL-3, and 0.3 ng/ml IL-5; each value represents 1 unit of that substance.
Radiolabeling Cytokines-- Recombinant IL-3 and GM-CSF were radioiodinated by the iodine monochloride method (24) to a specific activity of about 36 mCi/mg. Routinely, 4 µg of protein was iodinated and separated from iodide ions on a Sephadex G-25 column (Pharmacia Biotech Inc.), eluted with PBS containing 0.02% v/v Tween 20, and the iodinated proteins were stored at 4 °C for up to 4 weeks.
Saturation Binding Assays--
Binding assays were performed on
confluent monolayers in 24-well plates over a concentration range of 10 pM to 10 nM 125I-labeled GM-CSF or
IL-3 in binding medium (RPMI containing 0.5% (w/v) BSA/0.1% (w/v)
sodium azide) essentially as described previously (25). After
incubation at room temperature for 2 h, radioligand was removed,
and the cells were briefly washed twice in binding medium. Specific
counts were determined after lysis of the cell monolayer with
subsequent transfer and counting on a counter (Cobra Auto Gamma;
Packard Instruments Co., Meridien, CT). Dissociation constants were
calculated using the EBDA and LIGAND programs (26) (Biosoft, Cambridge,
United Kingdom).
Monoclonal Antibodies to the IL-3, GM-CSF, and IL-5
Receptors--
Monoclonal antibodies directed against GM-CSFR,
IL-3R
, IL-5R
, or
c were generated as described
previously (27) and purified and characterized as detailed elsewhere
(17, 18, 27). The monoclonal antibodies 8E4, 4H1, 9F5, and A14 were
selected for their ability to specifically immunoprecipitate
c, GM-CSFR
, IL-3R
, and IL-5R
, respectively. The
monoclonal antibody 1C1 conjugated to biotin was used for
immunoblotting
c, and 8E4, 4H1, 9F5, and A14 were used
for cell surface expression staining for
c, GM-CSFR
,
IL3R
, and IL5R
, respectively. The monoclonal antibodies were
purified from ascites as described (27). The monoclonal antibody (mAb)
against phosphorylated tyrosine was the peroxidase-conjugated
anti-phosphotyrosine 3-365-10 (Boehringer Mannheim).
Analysis of Receptor Cell Surface Expression by Flow
Cytometry--
Cell surface expression of transfected receptor
subunits was confirmed by indirect immunofluorescence staining using
anti-receptor and
chain specific monoclonal antibodies.
Staining was performed as described previously (17) and analyzed with a
EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL).
Surface Labeling of Cells and Immunoprecipitation-- COS cells were cell surface-labeled with 125I by the lactoperoxidase method as described previously (28). Approximately 108 cells were washed twice in PBS and then labeled with 1 mCi of 125I (NEN Life Science Products and AMRAD Pharmacia) in PBS. Cell were lysed in lysis buffer consisting of 137 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10% glycerol, 1% Nonidet P-40 with protease inhibitors (10 µg/ml leupeptin, 2 mM phenylmethlysulfonyl fluoride, 10 µg/ml aprotinin), and 2 mM sodium vanadate for 30 min at 4 °C followed by centrifugation of the lysate for 15 min at 12,000 g 4 °C. Following a 1-h preclearance with protein A-Sepharose (Pierce) at 4 °C, the supernatant was incubated for 18 h with 5 µg/ml antibody. Protein-immunoglobulin complexes were captured by incubation for 1 h with protein A-Sepharose followed by six subsequent washes in lysis buffer. Samples were boiled for 10 min in SDS sample load buffer either in the presence or absence of 2-mercaptoethanol (i.e. reducing or nonreducing) before separating immunoprecipitated proteins by SDS-PAGE. Immunoprecipitation from HEK293T cells was carried out similary except the cells were not surface labeled.
SDS-Polyacrylamide Gel Electrophoresis-- Immunoprecipitated proteins were analyzed by SDS-PAGE on polyacrylamide gels. Samples were boiled in SDS loading buffer for 5 min prior to loading. Molecular weights were estimated using SeeBlueTM prestained standards (Novex French's Forest, New South Wales, Australia). Radiolabeled proteins were visualized using an ImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Immunoblot and ECL--
Immunoprecipitated proteins separated by
SDS-PAGE were transferred to nitrocellulose membrane by
electroblotting. Routinely, nitrocellulose membranes were blocked in a
solution of PBS/0.05% (v/v) Tween 20 containing 1% (w/v) blocking
reagent 1096 176 (Boehringer Mannheim) and probed with either
peroxidase-conjugated anti-phosphotyrosine 3-365-10 (Boehringer
Mannheim) or anti-c (1C1-biotin) followed by
Streptavidin-POD (Boehringer Mannheim). Immunoreactive proteins were
detected by chemiluminescence using the ECL kit (Amersham Corp.)
following the manufacturer's instructions.
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RESULTS |
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Rationale for Mutagenesis of N-terminal Cysteine Residues of
c--
To study the molecular events involved in the
activation of the IL-3, GM-CSF, and IL-5 receptors, we replaced several
extracellular cysteine residues of
c by alanine
residues. So as to target Cys available for intermolecular
interactions, we sought to avoid Cys involved in structurally important
intramolecular disulfide bonds. By homology with other cytokine
receptors, domains one and three are expected to possess two disulfide
bonds each. This is clearly the case with domain three, which contains
only four Cys residues. However, domain one
c possesses
seven Cys, of which only Cys-34, Cys-45, and Cys-75 could be aligned
readily with equivalent Cys residues in other receptors (Fig.
1). Of the remaining Cys residues,
Cys-86, Cys-91, and Cys-96 are conserved in murine
c. Of
these, Cys-96 is followed by an Ile at position 98, which aligns with
conserved hydrophobic residues in other cytokine receptors, suggesting
that this Cys is part of the second conserved disulfide bond. Cys-96
and Cys-91 are proposed to lie in an extended loop between the D and E
beta strands of the first domain. Although Cys-86 and Cys-91 were
favored candidates for intermolecular disulfide bond formation, we
chose to also mutate the nearby Cys-96 and Cys-100 and the single Cys
residue in domain 2 at position 234.
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Mutation of Cys-86 and Cys-91 Selectively Disrupt Ligand-induced,
Disulfide-linked Heterodimer Formation--
Expression plasmids
encoding IL-3R and wild type (wt) or C86A, C91A, C96A, C100A, and
C234A mutant
c were co-transfected into COS cells. After
48 h, the cells were 125I surface-labeled and either
left unstimulated or stimulated with IL-3. IL-3R
and
c were then immunoprecipitated with specific MAbs 9F5
and 8E4, respectively, and the proteins were resolved by 6% SDS-PAGE
under either nonreducing or reducing conditions. We found that the
c mutants C100A and C234A behaved very similarly to wt
c. Both mutants allowed the formation of two high
molecular weight complexes in response to IL-3 (Fig.
2A), which, as with wt
c, contain IL-3R
and
c (Ref. 17 and
data not shown). These two complexes were immunoprecipitated by both
anti-IL-3R
mAb 9F5 and anti-
c mAb 8E4 (Fig.
2A). In the absence of IL-3, the anti-IL-3R
mAb 9F5
immunoprecipitated only monomeric IL-3R
, whereas the
anti-
c mAb 8E4 immunoprecipitated monomeric
c as well as the high molecular weight complex
corresponding to disulfide-linked
c homodimers (4, 17)
(Fig. 2A). As with the disulfide-linked dimers, the
noncovalent IL-3R
and
c heterodimers were not
affected by mutating Cys-100 or Cys-234 because both the anti-IL-3R
mAb and the anti-
c mAb co-immunoprecipitated both
IL-3R
and
c, and they did so only in the presence of
IL-3 (Fig. 2B).
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The c Mutants C86A and C91A Do Not Disrupt IL-3 and
GM-CSF High Affinity Binding--
We next examined the ability of Cys
mutants of
c to support high affinity IL-3 or GM-CSF
binding. COS cells were transfected with IL-3R
and GM-CSFR
and
either wt
c or the Cys
Ala
c mutants
and subjected to saturation binding studies with 125I-IL3
and 125I-GM-CSF. Scatchard transformation of the saturation
binding curves were performed and the Kd and
receptor numbers determined using the Ligand program. We found that
c bearing Cys
Ala substitutions at positions 86, 91, 100, and 234 were able to form high affinity binding sites (Fig.
4 and Table
I). The range of affinities for IL-3 high
affinity binding of these Cys
Ala
c substitution mutants varied from 31 to 280 pM, compared with 330 pM for wt
c, whereas GM-CSF high affinity
binding ranged from 27 to 230 pM, compared with 120 pM for wild type
c. In contrast, COS cell transfectants expressing the C96A
c showed no detectable
high affinity binding (Fig. 4 and Table I).
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C86A and C91A Abolish IL-3-, GM-CSF-, and
IL-5-dependent Tyrosine Phosphorylation of
c--
It has been previously established that
stimulation of cells with IL-3 leads to the formation of
disulfide-linked heterodimers of IL-3 receptor
and
c
chain, which is associated with phosphorylation of
c
(17). Similarly, in the case of the GM-CSF receptor, receptor heterodimerization occurs upon stimulation with GM-CSF, and this is
accompanied by
c phosphorylation (4). Here we show that in addition to the IL-3 and GM-CSF receptors, the IL-5 receptor forms
disulfide-linked complexes that are similarly accompanied by
c phosphorylation (Fig.
5). The relative proportion of
phosphorylated
c in the disulfide-linked heterodimer and
in monomeric
c varied between the three receptors. This
may be due to kinetic differences in receptor assembly or in the
stability of each receptor heterodimer. We have now taken advantage of
the inability of
c mutants to form disulfide-linked
heterodimers to determine whether the formation of these is necessary
for receptor activation or whether noncovalent dimerization is
sufficient for activation as measured by
c
phosphorylation. We transfected wt
c and the different
c mutants in HEK293T cells together with JAK-2 and
either IL-3R
, GM-CSFR
, or IL-5R
chain cDNA. After 48 h, the cells were either not treated or treated with IL-3, GM-CSF, or
IL-5, lysed, and immunoprecipitated with mAb 8E4 anti-
c.
The immunoprecipitates were separated on SDS-PAGE gels under reducing
conditions and Western blotted with antiphosphotyrosine antibody.
Mutants C100A and C234A and wt
c, which heterodimerize with the receptor
chain in a disulfide-linked manner in response to
ligand, showed phosphorylation of
c. In contrast,
mutants C86A, C91A, and C96A, which have lost the ability to
heterodimerize in a disulfide-linked manner in response to
ligand, have lost the potential to be phosphorylated in response to
IL-3, GM-CSF, or IL-5 (Fig. 6).
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DISCUSSION |
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We show here that disulfide-linked heterodimerization of
the GM-CSF, IL-3, and IL-5 receptors is essential for receptor
activation by the cognate ligand. Furthermore, we have identified
Cys-86 and Cys-91 in the N-terminal domain of c as the
key Cys residues involved in heterodimerization with the
chain of
each receptor. Comparison with other cytokine receptors indicates that
these Cys residues constitute a conserved motif present only in human and mouse
c and in
IL-3, suggesting that
it subserves a specialized function restricted to the GM-CSF, IL-3, and
IL-5 receptor family.
We have previously shown that the human IL-3 and GM-CSF receptors
undergo both noncovalent and disulfide-linked dimerization upon ligand
binding (4). We have now extended these observations to the IL-5
receptor, demonstrating that disulfide-linked dimerization is a common
theme in this receptor subfamily. To identify the Cys residues in
c responsible for disulfide linkage with the IL-3,
GM-CSF, and IL-5 receptor
chains, we mutagenized five of the eight
Cys residues in the N-terminal CRM of
c that, from alignment with other cytokine receptors, represented the best candidates for intermolecular interactions. We found a range of sensitivities to mutation of these Cys residues that could be correlated with their interspecies and interreceptor conservation.
The first class of Cys residue is exemplified by Cys-100 and Cys-234.
These residues are not conserved with even the closely related mouse
chains and we found no phenotype on replacing them with alanine
residues. The second class is represented by Cys-96, which is
apparently a conserved residue in both the mouse
chains and the
cytokine receptor family at large (29) and is inferred to be involved
in a structurally conserved disulfide bond. This residue is apparently
required for the structural integrity of the first domain of
h
c if not the entire extracellular portion of the
molecule. Although the C96A mutation permitted cell-surface expression
of h
c, it did not support high affinity binding of GM-CSF or IL-3 despite the substitution being well removed in sequence,
and presumably spatially distant, from the fourth domain of the
receptor that encompasses the majority of the ligand-recognition determinants (18, 25). The exact molecular basis for this observation
is uncertain, but it may be related to sequestration of
h
c into very large aggregates (Fig. 2A) that
obscure the ligand-contact site.
The third and most interesting class of cysteine mutation is that of
C86A and C91A, analogues that have lost their ability to form
disulfide-linked heterodimers but still retain the ability to associate
noncovalently with the subunit upon stimulation with ligand (Fig.
2). Although these mutants exhibited some propensity to aggregate in
the absence of stimulation, they retain the ability to interact with
ligand as judged by their ability to support high affinity binding.
Importantly, these analogues are deficient in phosphorylation of
tyrosine residues in
c; however, receptor-mediated functions and downstream signaling remains to be ascertained.
The observation of identical phenotypes with either mutation C86A or
mutation C91A suggests that these residues may cooperate functionally
in the native receptor such as via formation of an additional
intramolecular disulfide. This is consistent with our molecular
modelling of c, which suggests that Cys-86 and Cys-91 are sufficiently close to allow the formation of such a disulfide bond.2 In the presence of
ligand, this bond is proposed to undergo disulfide exchange with a free
sulfhydryl group from the
chain that is brought into proximity via
ligand-dependent noncovalent association.
Previous experiments have noted a correlation between
disulfide-linked receptor dimerization and receptor activation.
IL-6 induces covalent dimerization of two molecules of gp130 (9), and
CNTF induces covalent dimerization of gp130 with the LIF receptor (10).
Similarly, IL-3 (Fig. 5A), GM-CSF (Fig. 5B), and
IL-5 (Fig. 5C) induce covalent dimerization of
c with the corresponding
chain. In all of these
cases, concomitant phosphorylation of the receptor has been observed;
however, a causal relationship has not been established. The use of the
C86A and C91A mutants allowed us to demonstrate that noncovalent
receptor associations are not sufficient for receptor tyrosine
phosphorylation and that this requires disulfide linkage of receptor
subunits. The role of covalent dimerization seems to be to ensure
concomitant dimerization of the cytoplasmic portions of
c, facilitating transphosphorylation of associated
kinases of the JAK family. Indeed, experiments using chimeric receptors
that cause artificial dimerization of the cytoplasmic portions of
c lead to their activation (30, 31), and direct dimerization of JAK has been shown to transduce a growth signal (32).
Although they are essential for normal activation (14-16), the role of
the cytoplasmic domains of the receptor
chains remains unclear;
however, they may serve to orientate to
chains so as to juxtapose
correctly the JAK molecules or to participate directly in certain
functions (33, 34).
The chain forms intermolecular disulfides specifically with the
chains of the GM-CSF, IL-3, or IL-5 receptors. A common feature of
these
chains is the presence of an N-terminal FnIII-like domain of
a type restricted to this subfamily of cytokine receptors. Within this
N-terminal domain, all three receptor
chains possess an uneven
number of Cys residues in the N-terminal domain, suggesting that these
Cys residues are the most likely candidates to act as partners for
c. The IL-5R
has only a single Cys residue in the
N-terminal domain, at position 86, and this residue has been shown to
be important for IL-5 binding to the receptor (35).
The stoichiometry of the IL-3, GM-CSF, and IL-5 receptor complexes is
not known. The formation of an intermolecular disulfide bond between
Cys-86 or Cys-91 of c and a Cys in the N-terminal domain
of a receptor
chain could potentially occur with either the
chain with which it shares ligand or a second
chain, recruited as
part of a hexameric complex, as seen with the IL-6 receptor (36).
Because the individual FnIII-like domains of the receptor
chains
and
c are likely to be fairly rigid units with a length of 3.5-4.5 nm, the ability of
c to contact
chain
will depend on the interdomain angles that they can adopt. The receptor
and
c chains are class 1 cytokine receptors, and the
angles observed between the two domains of the CRM in the known
structures of this family, growth hormone receptor (5) and
erythropoietin receptor (37) are approximately 90°. It is therefore
reasonable to infer that the angles between domains 1 and 2 and domains
3 and 4 of
c and between domains 2 and 3 of the receptor
chains will also be approximately 90°. The conformation of the
linker peptides between domains 2 and 3 of
c or between
domains 1 and 2 of the receptor
chains cannot be gauged by
reference to homologous structures. Even if the linker peptides
permitted the membrane-distal portions to fold back over the cytokine
binding portions of the receptors, this would be unlikely to facilitate
a sufficiently close approach of Cys residues to allow formation of the
observed intermolecular disulfide bonds. Rather, we propose that the
intermolecular disulfide bond forms between
c and an
chain from a second receptor heterodimer (Fig.
8) because this can be accommodated
readily with respect to both the sizes of the domains and their
interdomain angles.
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Based on the likely orientation of their N-terminal domains with
respect to the CRM of the chains, we favor a receptor complex arranged clockwise when viewed from outside the cell in the order
chain1/ligand1/
chain1S-S
chain2/ligand2/
chain2. The
disulfide linkage of
c in one receptor heterodimer to an
chain in a second receptor heterodimer would facilitate juxtaposing
two
c molecules with their associated JAK kinases and
induce receptor phosphorylation. This initial association may also
facilitate the formation of a second disulfide between
c
in receptor 2 and an
chain in receptor 1 (Fig. 8, A and
B). The formation of a 2:2:2 complex is also consistent with
the requirement of two
chains in an active ligand-receptor complex
(38). On the other hand, a 1:1:1 stoichiometry has been suggested from
experiments using chimeras of
and
c with fos and jun
leucine zippers, although the formation of higher order complexes was
not excluded (34). The direct measurement of
-
c
interactions in solution may ultimately resolve this question.
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ACKNOWLEDGEMENTS |
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We thank Anna Nitschke for excellent secretarial assistance and Dr. Michael Berndt for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council 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.
Florey Fellow of the Royal Adelaide Hospital Research Fund.
§ To whom correspondence should be addressed: The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, P. O. Box 14, Rundle Mall, Adelaide 5000, South Australia, Australia. Tel.: 61-8-8222-3471; Fax: 61-8-8222-3538; E-mail: alopez{at}immuno.imvs.sa.gov.au.
1
The abbreviations used are: IL, interleukin;
GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR,
GM-CSF receptor; IL-2R, IL-2 receptor; c, common
chain; CRM, cytokine receptor module; mAb, monoclonal antibody; PAGE,
polyacrylamide gel electrophoresis; wt, wild type; LIF, leukemia
inhibitory factor; CNTF, ciliary neutrotrophic factor; CNTFR, CNTF
receptor.
2 F. C. Stomski, J. M. Woodcock, B. Zacharakis, C. J. Bagley, Q. Sun, and A. F. Lopez, unpublished observations.
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REFERENCES |
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