From the Division of Molecular Bioscience, John
Curtin School of Medical Research and the § Research School
of Chemistry, Australian National University,
Canberra, ACT 0200, Australia
Received for publication, November 15, 2002
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ABSTRACT |
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The receptors for human interleukins 3 and
5 and granulocyte macrophage colony-stimulating factor are
composed of ligand-specific Granulocyte-macrophage colony stimulating factor
(GM-CSF),1 IL-5, and IL-3 are
three cytokines produced by activated T-cells during immune responses
that are important mediators of inducible hematopoiesis and
inflammation. They signal through a shared receptor (the The receptors for IL-5, IL-3, and GM-CSF consist of cytokine-specific
Structurally, the Our recent determination of the crystal structure of the complete
extracellular domain of the human -subunits and a common
-subunit
(
c), the major signaling entity. The way in which
c interacts
with ligands in the respective activation complexes has remained poorly
understood. The recently determined crystal structure of the
extracellular domain of
c revealed a possible ligand-binding
interface composed of domain 1 of one chain of the
c dimer and the
adjacent domain 4 of the symmetry-related chain. We have used
site-directed mutagenesis, in conjunction with ligand binding and
proliferation studies, to demonstrate the critical requirement of the
domain 1 residues, Tyr15 (A-B loop) and
Phe79 (E-F loop), in high affinity complex formation and
receptor activation. The novel ligand-receptor interface formed between
domains 1 and 4 represents the first example of a class I cytokine
receptor interface to be composed of two noncontiguous fibronectin III domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
c receptor)
and play an important role in the pathogenesis of allergic disorders
and inflammatory diseases of the lung, such as asthma (1, 2).
Eosinophilia is controlled primarily by IL-5 (3, 4) and to a lesser
extent by IL-3 and GM-CSF. GM-CSF and IL-3 are believed to be centrally
involved in other chronic inflammatory diseases, such as arthritis and
multiple sclerosis (5, 6).
receptors essential to the activation of the shared common
-receptor subunit (
c), which is believed to be the main signaling
entity (7-10). Human GM-CSF and IL-3 bind to their cognate
receptors with low affinities, but in the presence of
c, high affinity complexes are formed. For example, with GM-CSF, the
Kd values for low and high affinity binding are
2-10 nM and 50-100 pM, respectively. Human
IL-5 differs from IL-3 and GM-CSF in that it binds to its
receptor
(IL-5R
) with greater affinity, and there is only a small affinity
conversion by the
c subunit (10, 11). The formation of a complex
involving ligand and the
and
c receptors is necessary for
receptor activation and signaling. The cytoplasmic portions of the
and
c subunits possess no intrinsic tyrosine kinase activity (12)
but in the activated receptor complexes formed with all three ligands
interact with and activate Janus kinase 2 (13), leading to the
phosphorylation of eight tyrosine residues located in the
c
cytoplasmic domain (14, 15). Subsequently, several signaling pathways
are induced, including the Janus kinase/STAT, Ras/mitogen-activated
protein kinase, and phosphatidylinositol 3-kinase pathways (reviewed in
Ref. 16). The structures of the activation complexes involving the
c
receptor are unknown.
and
c subunits of the GM-CSF, IL-3, and IL-5
receptors belong to the cytokine class I receptor superfamily (or
hemopoietin receptor family), which includes the growth hormone, erythropoietin, gp130, and IL-4
receptors. The characteristic feature of this family is the extracellular cytokine receptor homology
module, composed of two fibronectin III domains that contain a number
of conserved sequence elements (17, 18). The crystal structures of the
receptor-ligand complexes of the growth hormone (19), erythropoietin
(20, 21), and IL-4
(22) receptors have been solved, and through
mutagenesis studies (23-27), the ligand-binding epitopes have been
elucidated. The extracellular domains of these receptors contain two
approximately orthogonal fibronectin III domains that each contribute
key residues for ligand binding from loops at the receptor "elbow"
region. To what extent these principles apply to the
c subunit has
not been clear, because
c does not bind any of its three ligands directly, has four fibronectin III domains in its extracellular region
rather than two, and heterodimerizes with the respective
receptors.
By analogy with the other class I receptors, several groups have
predicted the existence of an elbow region formed by domains 3 and 4 of
c that would serve as a ligand-binding interface. Mutagenesis
studies have been successful in identifying residues in the B'-C' (28,
29) and F'-G' (30) loops of domain 4 that are critical for high
affinity ligand binding, but no critical residues have been detected in
domain 3.
c subunit (Fig.
1) showed it to exist as a stable,
intertwined homodimer (32). A number of lines of evidence indicate that
the dimer is not confined to the crystal and is likely to be the
functional form of the receptor in vivo (32). A highly novel
feature of the structure is that strand G of domain 1 forms strand G of
domain 3 of the dimer-related molecule, and likewise strand G of domain
3 partners with the dimer-related domain 1. The unique interlocking
dimeric structure of
c results in the juxtapositioning of domain 1 of one chain adjacent to domain 4 of its dimer-related equivalent chain
(Fig. 1). The interface between domains 1 and 4 is approximately
orthogonal and resembles the ligand-binding interface of the growth
hormone, erythropoietin, and IL-4
receptors (Fig. 1). Thus, by
analogy with other class I cytokine receptors, domains 1 and 4 could
form a ligand-binding interface involved in high affinity complex
formation. However, it has not been clear to what extent the binding
interactions in the
c activation complex conform to the principles
for ligand binding already established in the cytokine class I receptor
family or whether novel types of interactions are involved.
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Fig. 1.
Structure of the c
receptor. Top panel, structure of the
c
homodimer. Chain A is yellow, and chain B is red.
Glycosylation is shown by sticks colored by atom type
(green, carbon; red, oxygen; and blue,
nitrogen). The domains of chain A are labeled in yellow
text, and those of chain B are in red text. The loops
of domains 1 and 4 potentially relevant to ligand-receptor interaction
are labeled in white text. Bottom left panel, the
IL-4·IL-4
receptor complex. IL-4 is green, and IL-4
receptor is orange. The loops of the IL-4
receptor
involved in complex formation are labeled. Bottom right
panel, the domain 1-domain 4 interface of
c, which resembles
the ligand-binding interface of the IL-4
receptor. The
c loops
equivalent to the IL-4
ligand-binding loops are labeled. The figures
were drawn using RIBBONS (31). The structures of
c and the IL-4:IL-4
receptor complex are taken from Protein Data Bank codes 1gh7 and
1iar, respectively.
In the present work, we have used site-directed mutagenesis, ligand
binding, and proliferation assays to establish that Tyr15
and Phe79 of domain 1 of c are required for high
affinity ligand binding and receptor activation. The novel
ligand-receptor interface, which involves cooperation between domains 1 and 4 of two different chains of the
c receptor dimer, is the first
example in the class I cytokine receptor family of a functional epitope
that involves noncontiguous fibronectin III domains.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis of Human c cDNA--
A cDNA
encoding the extracellular domain of the human
c subunit (residues
1-443) was cloned from HL60 eosinophils (33). A full-length cDNA
was constructed by substituting a BstXI-BssHII fragment of the extracellular domain of the cDNA for
c isolated from TF1 cells (8). Site-directed mutagenesis was carried out using the
QuikChange method (Stratagene, La Jolla, CA) with Pfu Turbo
DNA polymerase. The complete sequences of mutant
c cDNAs were
verified by Big Dye Terminator cycle sequencing (Applied Biosystems,
Gladesville, Australia).
Expression Constructs--
For expression in COS7 cells, the
cDNAs encoding human GM-CSFR (from T. Willson, Walter and Eliza
Hall Institute, Melbourne, Australia) or IL-5R
and wild type or
mutant
c subunits were subcloned into pCEX-V3-Xba, a vector derived
from pCEX-V3 (34). For expression in CTLL-2 cells, cDNAs encoding
human GM-CSFR
or IL-5R
were subcloned into pEF-IRES-N, and the
c subunits were subcloned into pEF-IRES-P (35) (from S. Hobbs,
Institute of Cancer Research, London, UK).
Cytokines and Radiolabeling--
Human IL-5, human GM-CSF, and
murine IL-2 were produced using the baculovirus expression system (33).
Purified IL-5 was radiolabeled as described previously (36).
Radiolabeled IL-5 was stored at 4 °C and used for up to 7 days.
Radiolabeled GM-CSF was purchased from PerkinElmer Life Sciences,
stored at 70 °C, and used for up to 6 weeks.
Cells and DNA Transfections--
COS7 cells were maintained in
Dulbecco's modified Eagle's medium containing 10% bovine serum and
CTLL-2 cells as described previously (33). DNA expression constructs
were introduced into cells by electroporation (33). COS7 cells were
harvested using trypsin and electroporated with 10 µg of GM-CSFR
or IL-5R
and 25 µg of wild type or mutant
c constructs at 200 V
and 960 microfarads. Binding studies and antibody staining were
performed on cells 64-68 h after transfection. CTLL-2 cell lines
stably expressing GM-CSFR
or IL-5R
and wild type or mutant
c
subunits were generated in two steps, as described previously (33).
Equilibrium Binding Analysis for GM-CSF and IL-5--
COS7 cells
expressing the relevant receptors were used to study IL-5 and GM-CSF
binding and were harvested as described previously (28). CTLL-2 cells
expressing the IL-5R and
c subunits were cultured in IL-5 and
washed four times with factor-free basal medium before use in binding
assays. 106 cells in resuspended binding medium (RPMI 1640 supplemented with 10 mM HEPES and 0.5% w/v bovine serum
albumin) were incubated with increasing concentrations of radioligand
for 2-3 h at 4 °C. Nonspecific binding was determined by the
addition of a 100-fold excess of unlabeled ligand to samples in which
high radioligand concentrations were added. The nonspecific binding was
interpolated for lower radioligand concentration data using linear
regression. The assays were terminated by centrifugation of each
binding solution through 0.2 ml of 2:1 v/v dibutyl phthalate:dinonyl
phthalate at 12,000 × g for 4 min (37). The tips of
tubes, containing the visible cell pellet and associated
radio-iodinated ligand, were counted using a Packard 5780 Auto-gamma
counter. The dissociation constants (Kd) and
Scatchard transformations were calculated from specific binding data
using the EBDA (38) and LIGAND (39) programs (Biosoft, Cambridge, UK).
Multiple data files were co-analyzed to obtain more accurate estimates
of Kd values. One- and two-site models were
evaluated in LIGAND and only when statistically significant
(p < 0.05), a two-site model was used to determine Kd values.
Flow Cytometry--
COS7 cells transfected with cDNAs
encoding wild type or mutant c subunits were incubated in the
presence of rabbit antiserum raised against the
c(HL60)
extracellular domain for 30min at 4 °C. The cells were washed and
incubated with a fluorescein isothiocyanate-conjugated anti-rabbit
secondary antibody (Sigma) for 25 min at 4 °C and then washed before
analysis of cell surface expression was performed on a FACScan flow
cytometer (Becton Dickinson, North Ryde, Australia).
Proliferation Assays--
[3H]Thymidine
incorporation assays were performed as described previously (33) to
determine the capacity of the wild type or mutant c subunits to
deliver a proliferative signal in CTLL-2 stable cell lines.
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RESULTS |
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The Involvement of Domain 1 of c in Ligand Binding--
In the
well established class I cytokine receptor superfamily ligand-binding
motif, critical ligand-binding residues are commonly contributed from
the E-F and A-B loops of the membrane-distal fibronectin III domain and
from the B'-C' and F'-G' loops of the membrane-proximal domain (Fig.
1). From the x-ray structure of
c (Fig. 1), it is clear that if
ligand-binding with
c follows the previously established principles,
the interface would involve residues from loops in domain 1 cooperating
with those from domain 4 of the partner molecule of the
c dimer. The
loops of domain 1 that could potentially be involved in ligand binding
are the A-B, E-F, and C-D loops. Site-directed mutagenesis of the human
c cDNA was therefore performed to generate alanine mutants of the residues in these loops. In studies on cytokine class I receptors, alanine substitution is commonly used for the determination of residues
whose side chains are involved in ligand binding. Alanine has a
hydrophobic side chain but is often found in solvent-exposed regions of
proteins, and its introduction does not disrupt the geometry of the
main-chain, nor does the methyl side chain cause electrostatic or
steric interference. Because GM-CSF has readily distinguishable low and
high affinity binding, we used this ligand as a model to test the
binding of the mutants. We also examined the binding of IL-5, because
this cytokine, unlike GM-CSF, is an interlocking dimer and could
potentially interact with
c in a different way.
GM-CSF Binding Properties of Wild Type and Mutant c
Receptors--
COS7 cells were co-transfected with expression vectors
encoding human GM-CSFR
and wild type or mutant
c subunits.
Saturation binding assays were then performed on the transiently
transfected COS7 cells using 125I-labeled human GM-CSF, and
the dissociation constants (Kd) were determined
(Table I).
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COS7 cells transfected with the cDNA encoding GM-CSFR
were found to bind GM-CSF with low affinity (Kd, 8.5 nM). Cells expressing both wild type
c and GM-CSFR
exhibited two GM-CSF-binding sites, as illustrated by the curvilinear
Scatchard plot (Fig. 2). The
Kd values of 58 pM (high affinity) and
8.3 nM (low affinity) are consistent with those previously reported for the GM-CSF receptor system (8, 28-30). The wild type
c
receptor used in this work and in the x-ray structural studies has a
six-amino acid insertion in the C-D loop of domain 3. This
c variant
is the major form in HL60 eosinophils and appears to be a splice
variant with normal function (33). It has not previously been examined
in binding studies. The Kd values obtained indicate
that this variant has normal binding properties. We also included as a
control a mutant (Y403A) affected in one of the key
ligand-binding residues in domain 4. In agreement with previous studies
(30, 40), mutation of Tyr403 was found to abolish high
affinity binding (Table I).
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Involvement of Loops in Domain 1 of the c Subunit in GM-CSF
Binding--
The first loop of domain 1 to be examined was the A-B
loop. This loop consists of residues Tyr15,
Thr16, Ser17, and His18. Each
residue was individually mutated to alanine, and the resultant mutants
were examined for GM-CSF binding. No high affinity GM-CSF receptors
were detected on cells co-transfected with cDNAs encoding GM-CSFR
and the mutant
c subunit Y15A (Table I and Fig. 2). This
result indicates that Tyr15 is a critical binding
determinant in the high affinity binding of GM-CSF. The Scatchard plot
of GM-CSF binding with Y15A
c indicates the presence of only low
affinity sites. No other residues located in the A-B loop were found to
be essential for high affinity binding (Table I). It was also of
interest to determine whether phenylalanine could substitute for
tyrosine in GM-CSF binding, and therefore the mutant
c, Y15F, was
prepared. Y15F was found to give normal high affinity binding (Table
I), indicating that the hydroxyl group of Tyr15 is not
involved and that Phe is an effective substitution for Tyr at this position.
We next examined the involvement of the C-D and E-F loops of domain 1 in GM-CSF binding. The C-D loop consists of residues Asn42,
Glu43, Asp44, Leu45, and
Leu46. A mutant was generated in which all of the C-D loop
residues were mutated to alanine. This mutant gave normal high affinity binding, indicating that the C-D loop is not involved (Table I). The
E-F loop is made up of residues Cys76, Gln77,
Ser78, Phe79, Val80,
Val81, and Thr82. Mutation of the first three
residues in the loop to alanine did not affect high affinity GM-CSF
binding. The last four residues were individually mutated to alanine.
Of these, only Phe79 was found to be required for high
affinity GM-CSF binding. The F79A mutation of c completely abolished
high affinity binding (Table I and Fig. 2), indicating that the E-F
loop also plays an important role in forming the high affinity GM-CSF complex.
Cell Surface Expression of Y15A and F79A Mutant c
Receptors--
To verify that the loss of high affinity binding when
the Y15A and F79A
c mutants and GM-CSFR
were co-expressed did not result from a perturbation of the receptor translation, folding, or
presence on the cell surface, flow cytometry was used to detect
c
cell surface expression. COS7 cells transiently expressing wild type or
mutant
c subunits were stained with rabbit antiserum raised against
the human
c(HL60) extracellular domain and a fluorescein isothiocyanate-conjugated secondary antibody and examined by indirect flow cytometry (Fig. 3). The antiserum
used was shown to immunoprecipitate human
c from CTLL-2
GM-CSFR
/
c cell lysates, and the affinity of the antiserum for the
human
c extracellular domain was verified using an enzyme-linked
immunosorbent assay.2 The
level of cell surface expression of the Y15A and F79A mutant
c
subunits was observed to be equivalent to wild type
c expression with only small variations observed because of differences in transfection efficiency.
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Growth Responses of the Mutant c Receptors to
GM-CSF--
Although the formation of a high affinity complex is
believed to be a prerequisite for receptor activation and downstream signaling, we considered it important to correlate the binding data
with an unequivocal signaling response, in this case proliferation. The
GM-CSFR
cDNA was installed in the murine
IL-2-dependent lymphoid cell line, CTLL-2, using the
pEF-IRES-N vector, which encodes G418 resistance. G418-resistant cells
were subsequently transfected with wild type or mutant
c cDNAs
in the vector pEF-IRES-P, which encodes puromycin resistance. Following
selection with these antibiotics, resistant cells, which had been
maintained in the presence of murine IL-2, were used in proliferation
assays to assess their responsiveness to GM-CSF after murine IL-2 was
removed. The dose of GM-CSF required for half-maximal stimulation of
each cell line (ED50) was determined by GM-CSF titration.
The amount of GM-CSF giving 50% of maximal stimulation of the CTLL-2
cell line co-expressing the wild type
c and GM-CSFR
subunits was
defined as 1 unit, and the relative amounts of GM-CSF required for 50%
stimulation of cell lines expressing mutant receptors were determined.
Duplicate CTLL-2 cell lines co-expressing the Y15A or F79A mutant
c
and GM-CSFR
subunits were generated from separate transfections. The
transfected cells expressing the mutant receptors exhibited severely
reduced responsiveness to GM-CSF compared with cells expressing the
wild type receptor (Table II). The amount
of GM-CSF for a 50% growth response was increased 55-85-fold for Y15A
and 8-16-fold for F79A. The parent cell line, CTLL-2 GM-CSFR
, did not respond detectably to GM-CSF, even at doses in excess of 2000 ED50 units, verifying the absolute requirement of
c for
the growth response.
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Role of Tyr15 and Phe79 in High Affinity
Binding and Signaling with IL-5--
Because IL-5, unlike GM-CSF, is a
dimer, it was of interest to determine whether the same residues that
were critical for high affinity GM-CSF binding were also critical for
IL-5 binding. Human IL-5 binds to its cognate receptor with
unusually high affinity. Reported increases in binding in the presence
of the
c subunit range from 2- to 3-fold (10, 11, 29). We compared the binding of IL-5 to IL-5R
in the presence and absence of
c in
COS7 cells and found an increase in binding of less than 1.5-fold in
the presence of
c (Table III). CTLL-2
cells co-expressing the IL-5R
and
c subunits that gave a normal
growth response to IL-5 exhibited the same Kd as
COS7 cells co-expressing the IL-5R
and
c subunits (Table III).
The similarity of the dissociation constants observed for high and low
affinity IL-5 binding made it difficult to reliably distinguish IL-5
binding to the IL-5R
:
c complex from binding to IL-5R
alone.
However, we were able to establish that even though only a small
affinity conversion was detectable, IL-5 binding induced receptor
activation, because CTLL-2 cells co-expressing
c with the IL-5R
showed a growth response not seen with cells expressing IL-5R
alone
(Table II). We therefore examined the roles of Tyr15 and
Phe79 of the
c subunit in the proliferative response to
IL-5 in CTLL-2 cells. Separate transfection experiments were performed
to generate duplicate CTLL-2 cell lines co-expressing IL-5R
and wild
type, Y15A, or F79A
c subunits. The duplicate CTLL-2 cell lines
co-expressing the wild type
c and IL-5R
subunits gave similar
growth responses to IL-5, giving 50% of maximal stimulation with 1 and
1.93 units of IL-5, respectively. In contrast, cells expressing the
mutant
c subunit Y15A together with IL-5R
gave no detectable
growth response to IL-5, whereas cell lines expressing F79A together with IL-5R
required 7-15-fold more IL-5 for a 50% growth response than CTLL-2 cells co-expressing IL-5R
and wild type
c (Table II).
The parent cell line, CTLL-2 IL-5R
, did not detectably respond to
IL-5 doses in excess of 250 units. It is interesting that the effect of
the two mutations on the growth responses to both GM-CSF and IL-5 was
very similar, with the Y15A mutant being more severely affected in
growth signaling than the F79A mutant, even though the loss of high
affinity GM-CSF binding seemed comparable in both cases.
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DISCUSSION |
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To date, the definition of the ligand-binding interfaces in the cytokine class I receptor family has been accomplished primarily for two-domain receptors, like the growth hormone receptor, that bind their ligands directly. In these receptors, a conserved motif consisting of an approximately orthogonal elbow between the two fibronectin III domains serves as the ligand-binding interface. X-ray crystallography of ligand-receptor complexes has demonstrated that the structural epitopes for these receptors comprise a relatively large number of residues at the protein-protein interfaces. However, mutagenesis has shown that only a small subset of these residues (forming the functional epitope) play key roles in ligand binding. The functional epitopes determined so far involve at least one residue in each of the two fibronectin III domains comprising the interface, with the residues being located in the A-B or E-F loops of the membrane-distal domain and the B'-C' or F'-G' loops of the membrane-proximal domain. The key residues are clustered in a binding "hot spot."
In contrast to the two-domain receptors, much less is known about the
mechanism of ligand binding for a receptor like the c receptor that
consists of four fibronectin III domains and forms a high affinity
complex only with the ligands bound to their specific
receptors.
Previous work established the involvement of residues in the B'-C' (28,
29) or F'-G' (30) loops of the membrane-proximal domain (domain 4) but
unexpectedly no residues from the predicted membrane-distal domain
(domain 3). Our recent determination of the crystal structure of the
c extracellular domain revealed that
c exists as a homodimer with
domain 1 of one chain positioned adjacent to domain 4 of the other
chain (32), suggesting the possibility that the functional epitope for
high affinity ligand binding may involve domains 1 and 4. Indeed, we show here that the domain 1 residues, Tyr15 (A-B loop) and
Phe79 (E-F loop), are critical for the formation of the
high affinity GM-CSF complex and subsequent activation of human GM-CSF
receptor, as measured by a proliferative response. They are also
critical for the growth response to IL-5, another of the ligands that
shares the
c receptor subunit, even though IL-5 differs from GM-CSF in that it is a dimer.
CTLL-2 cells co-expressing the Y15A or F79A mutant c subunits and
the appropriate
receptors showed substantially diminished growth
responses to GM-CSF and IL-5. The Y15A mutant showed no detectable
growth response to IL-5 and a severely reduced response to GM-CSF. The
F79A mutant was less severely affected, but the data clearly indicate
the involvement of Phe79 in the growth response to the two
ligands. It is interesting that the Y15A and F79A
c mutants can
still signal detectably with high concentrations of GM-CSF despite
their inability to support high affinity binding of GM-CSF. This
finding is similar to the observation that the murine GM-CSF E21A
mutant is capable of stimulating the murine GM-CSF receptor at elevated
ligand concentrations, despite its inability to form a high affinity
complex (41). The detectable growth response of the mutant
c
receptors confirms their cell surface expression, in agreement with the
fluorescence-activated cell sorter analysis, indicating that single
amino acid substitutions did not disrupt their translation, folding, or
transport to the membrane.
The two critical residues identified in this study, Tyr15
and Phe79, are in the A-B and E-F loops of domain 1 of
c, respectively. Together with the previously identified residues in
domain 4 (Tyr347, His349, and
Ile350 in the B'-C' loop and Tyr403 in the
F'-G' loop), they form a functional epitope that resembles those of the
simpler two-domain cytokine class I receptors, such as the growth
hormone receptor. However, the
c receptor functional epitope is
novel because it is composed of domains contributed from two different
protein chains.
The c functional epitope shows a superficial similarity to cluster I
of the IL-4
receptor, which also features three tyrosines contributed by the A-B, B'-C', and F'-G' loops that are critical for
ligand-binding (22, 27). Additionally, like IL-4, GM-CSF, IL-3, and
IL-5, each possesses a conserved glutamic acid residue on the A-helix
that is critical for the interaction with
c (42-45). Despite
Tyr15 of
c being in a position analogous to the critical
Tyr13 of the IL-4
receptor, this study has illustrated
that the phenolic hydroxyl group of Tyr15 in
c is
dispensable for high affinity GM-CSF binding, whereas the phenolic
hydroxyl of Tyr13 is essential for IL-4
ligand binding
(27). This suggests that these two receptors engage their ligands by
different mechanisms.
The residues in the receptor hot spot of c form a less compact
cluster than those of the two-domain cytokine receptors, raising the
interesting possibility that
c functional epitope residues (for
example Tyr403) could be involved in contacts with the
subunits. Of the
c functional epitope residues, Tyr15,
Phe79, and Ile350, in contrast to
Tyr347, His349, and Tyr403, are not
highly exposed in the
c structure. The low solvent accessibility of
these residues, of itself, does not preclude a role in direct ligand
interaction because it is known that Phe205, a residue
critical for ligand binding by the erythropoietin receptor, is also not
highly exposed (26). In addition, ligand-induced receptor plasticity
has been well documented (46). However, it is also possible that the
role of Phe79 in
c could be to maintain the conformation
of Tyr15, because the two residues make interatomic
contacts in the
c structure.
There is relatively little information available regarding the key
residues involved in c recognition by the ligands, GM-CSF, IL-3, and
IL-5. To date only a single residue (Glu21 of GM-CSF,
Glu22 of IL-3, or Glu13 of IL-5) has been
identified as critical for interaction with the
c subunits (42-45).
In comparison, IL-5 has been shown by mutagenesis to interact with
IL-5R
via several residue side chains (45, 47).
Similarly, clusters of several residues on other cytokines have been
shown to be critical for receptor binding (48, 49). Thus, we would
expect that additional residues in the ligands that are involved in
c recognition remain to be discovered.
Although a detailed understanding of receptor activation must await the
determination of the structures of the respective activation complexes,
the present work provides compelling evidence for the existence of a
novel ligand-receptor interface in c formed by domains 1 and 4 and
identifies Tyr15 and Phe79 in domain 1 as
residues essential for
c function.
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ACKNOWLEDGEMENTS |
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We thank T. Willson for providing the
GM-CSFR cDNA, S. Hobbs for the pEF-IRES vectors, A. Church for
the purification of IL-5, I. Walker for the purification of GM-CSF, and
S. Gustin for preparation of the anti-
c serum.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Div. of Molecular Bioscience, John Curtin School of Medical Research, Canberra, ACT 0200, Australia. Tel.: 61-261252439; Fax: 61-261250415; E-mail: Ian.Young@anu.edu.au.
Published, JBC Papers in Press, January 12, 2003, DOI 10.1074/jbc.M211664200
1
The abbrieviations used are: GM-CSF,
granulocyte-macrophage colony-stimulating factor; IL, interleukin;
GM-CSFR,
subunit of GM-CSF receptor; IL-5R
,
subunit of
IL-5 receptor;
c, common
-subunit of GM-CSF, IL-3 and IL-5
receptors; STAT, signal transducers and activators of transcription.
2 S. E. Gustin, J. M. Murphy, and I. G. Young, unpublished results.
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