Biochem. J. (2010) 426, 307–317 (Printed in Great Britain)
307
doi:10.1042/BJ20091745
The Ig-like domain of human GM-CSF receptor α plays a critical role in
cytokine binding and receptor activation
Shamaruh MIRZA*, Andrew WALKER*, Jinglong CHEN*, James M. MURPHY†1 and Ian G. YOUNG*1
*Structural Biology Program, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia, and †Molecular Medicine Division, The Walter
and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia
GM-CSF (granulocyte/macrophage colony-stimulating factor)
is an important mediator of inducible haemopoiesis and
inflammation, and has a critical role in the function of alveolar
macrophages. Its clinical applications include the mobilization
of haemopoietic progenitors, and a role as an immune stimulant
and vaccine adjuvant in cancer patients. GM-CSF signals via a
specific α receptor (GM-CSFRα) and the shared hβc (human
common β-subunit). The present study has investigated the role
of the Ig-like domain of GM-CSFRα in GM-CSF binding and
signalling. Deletion of the Ig-like domain abolished direct GMCSF binding and decreased growth signalling in the presence of
hβc. To locate the specific residues in the Ig-like domain of GMCSFRα involved in GM-CSF binding, a structural alignment was
made with a related receptor, IL-13Rα1 (interleukin-13 receptor
α1), whose structure and mode of interaction with its ligand has
recently been elucidated. Mutagenesis of candidate residues in
the predicted region of interaction identified Val51 and Cys60 as
having critical roles in binding to the α receptor, with Arg54 and
Leu55 also being important. High-affinity binding in the presence
of hβc was strongly affected by mutation of Cys60 and was
also reduced by mutation of Val51 , Arg54 and Leu55 . Of the four
key residues, growth signalling was most severely affected by
mutation of Cys60 . The results indicate a previously unrecognized
role for the Ig-like domain, and in particular Cys60 , of GM-CSFRα
in the binding of GM-CSF and subsequent activation of cellular
signalling.
INTRODUCTION
The receptors for IL-5, IL-3 and GM-CSF consist of cytokinespecific α receptors essential to the activation of hβc, which is
believed to be the main signalling entity [6–9]. Human GM-CSF
and IL-3 bind to their cognate α receptors with low affinities
(1–50 nM) but, in the presence of hβc, high-affinity complexes
are formed (30–200 pM). By convention, the terms ‘low’ and
‘high’ affinity are used to distinguish the two receptor-binding
modes in studies of cytokine–receptor interactions, even though
in many areas of biochemistry the nanomolar affinity associated
with ‘low’-affinity binding would be considered very high. The
formation of a complex involving ligand, α and βc receptors is
necessary for receptor activation and signalling. The cytoplasmic
portions of α and βc subunits possess no intrinsic tyrosine kinase
activity [10], but the activated receptor complexes formed with
all three ligands interact with and activate JAK2 (Janus kinase 2)
[11], leading to the phosphorylation of eight tyrosine residues
located in the βc cytoplasmic domain [12,13]. Subsequently,
several signalling pathways are induced [14].
Structurally, the α and βc subunits of hGM-CSFR (human GMCSFR), hIL-3R (human IL-3 receptor) and hIL-5R (human IL5 receptor) belong to the cytokine class I receptor superfamily
(or haemopoietin receptor family), which includes GHR [GH
(growth hormone) receptor], the EPO (erythropoietin) receptor,
gp130 and IL-4R (IL-4 receptor)/IL-13R (IL-13 receptor). The
characteristic feature of this family is the extracellular cytokinereceptor homology module (CRM or CRH), composed of two
fibronectin III domains that contain a number of conserved
sequence elements (reviewed in [15]). Crystal structural analyses
GM-CSF (granulocyte/macrophage colony-stimulating factor),
interleukin (IL)-5 and IL-3 are three cytokines produced by
activated T-cells during immune responses that are important
mediators of inducible haemopoiesis and inflammation. They
signal through a shared receptor {hβc [human βc (common
β-subunit)]} and play an important role in the pathogenesis of
allergic disorders and inflammatory diseases of the lung, such as
asthma. Eosinophilia is controlled primarily by IL-5 [1,2] and, to
a lesser extent, by IL-3 and GM-CSF. IL-3 plays a critical role in
basophil and mast cell responses in parasite infections [3]. GMCSF is believed to be centrally involved in chronic inflammatory
diseases, such as arthritis and multiple sclerosis [4]. Of the three
cytokines, GM-CSF has received the most attention in terms of
clinical applications. GM-CSF has been applied to the supportive
care of cancer patients and, more recently, to the mobilization
of progenitors. The ability of GM-CSF to stimulate cytotoxic
immune responses gives it great potential as an immune stimulant
and vaccine adjuvant in cancer patients [4]. GM-CSF is also an
essential factor in maintaining alveolar homoeostasis through
its action on alveolar macrophages, and mutations affecting
GM-CSFRα (GM-CSF receptor α subunit) have recently been
detected in patients with pulmonary alveolar proteinosis [5]. A
critical first step in GM-CSF signalling involves its binding to the
specific GM-CSFRα. In the present study, we have demonstrated
an important role of the first domain of GM-CSFRα in binding
GM-CSF, an aspect that has not been studied previously.
Key words: common β-subunit (βc), cytokine receptor,
cytokine-receptor homology module (CRM), dissociation
constant, granulocyte/macrophage colony-stimulating factor
(GM-CSF), immunoglobulin-like domain, interleukin-3 (IL-3),
interleukin-5 (IL-5).
Abbreviations used: (h)βc, (human) common β-subunit; CRM, cytokine-receptor homology module; EPO, erythropoietin; FBS, fetal bovine serum; GH,
growth hormone; GHR, GH receptor; (h)GM-CSF, (human) granulocyte/macrophage colony-stimulating factor; (h)GM-CSFR, (human) GM-CSF receptor;
IL, interleukin; IL-3R, IL-3 receptor; (h)IL-5R, (human) IL-5 receptor; IL-6R, IL-6 receptor; (h)IL-13R, (human) IL-13 receptor; D1, the N-terminal Ig-like
domain of IL-13Rα1, GM-CSFRα, IL-5Rα or IL-3Rα, α subunits of GM-CSFR, IL-5R or IL-3R respectively; K d , dissociation constant; mIL-2, murine IL-2.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
308
S. Mirza and others
of the ligand–receptor complexes of GH, EPO, IL-4, IL-6,
IL-12, G-CSF (granulocyte colony-stimulating factor) and, very
recently, IL-13 have demonstrated that the extracellular domains
of these receptors possess CRMs consisting of two approximately
orthogonal fibronectin III domains that each contributes key
residues for ligand binding from loops at the receptor ‘elbow’
region [16–21].
In 2001, we reported the crystal structure of the ectodomain of
βc of the hGM-CSFR which showed that it was a radical departure
from the GHR paradigm [22]: being a pre-formed intertwined
dimer with novel chain swapping between domains. Subsequent
mutagenesis elucidated the functional epitope and showed that
the βc possessed an unusual ligand-binding elbow region formed
by the two protein chains [23,24]. GM-CSFRα together with
IL-3Rα and IL-5Rα is part of a sub-group of the class I cytokine
receptor superfamily which possess an additional ‘Ig-like’ domain
(D1) at the N-termini of their ectodomains in addition to the
CRM. In other cytokine receptor systems, structural studies have
clearly established the pivotal roles that Ig-like domains play in
mediating cytokine binding and receptor activation. For example,
the crystal structure of the IL-6–IL-6Rα (IL-6 receptor α subunit)–
gp130 complex demonstrates that the Ig-like domain of gp130
has a bridging function, interacting with the binding epitope
on IL-6 to recruit another trimer, to generate the hexameric
signalling complex [25]. Very recently, IL-13Rα1 D1 was shown
to play a key role in ligand-binding by engaging the dorsal
surfaces of IL-4 and IL-13 in their respective crystal structures
[26]. In the case of IL-5, alanine scanning mutagenesis of the
IL-5Rα subunit identified the residues Asp55 , Asp56 and Tyr57 in
IL-5Rα D1 as essential determinants of IL-5 binding [27,28].
Additionally, deletion of D1 of the hIL-5Rα (human IL-5Rα)
blocks IL-5 signalling [27]. A different situation exists for
IL-3Rα. We have demonstrated the existence of an isoform
lacking D1 which, although it does not bind IL-3 as tightly as
the full-length receptor, is competent for signalling [29].
In the case of hGM-CSF (human GM-CSF), whether D1 of
the GM-CSFRα subunit has a role in ligand binding and receptor
activation has not been established. Several studies have been
performed on the CRM of hGM-CSFRα to identify the functional
determinants involved in the GM-CSF–GM-CSFRα interaction,
the first step in receptor activation. Guided by homology
modelling with the GHR complex, Arg280 , from the F –G
loop of GM-CSFRα, was identified as being involved in ligand
binding [30]. A recent crystal structure of hGM-CSF in complex
with the ectodomains of the GM-CSFRα and βc subunits has been
an interesting new development in structural studies of the GMCSFR, and the authors have suggested that the active complex
is a dodecamer [31]. The new structure has provided valuable
verification of the previously determined βc structure [22] and
its functional epitope [23], and showed that the loop residues
241–251 and 299–305 of GM-CSFRα interact with residues 11–
23 (helix A) and residues 112–118 (helix D) of the cytokine
[31]. Unfortunately, the electron density for D2 was relatively
poor and no electron density was present for D1 of GM-CSFRα.
The structure therefore has provided no insights into (i) whether
domain D1 plays a role in GM-CSF recognition; and (ii) whether
this Ig-like domain participates in the signalling complex.
In the present study, we demonstrate the critical importance of
hGM-CSFRα D1 for GM-CSF binding and signal transduction.
To aid in the investigation of the role of the Ig-like domain we
prepared a structural homology model between hGM-CSFRα
and IL-13Rα1. Twelve residues in the probable region of
interaction with GM-CSF were mutated using alanine substitution
mutagenesis. This enabled us to identify the critical determinants,
Cys60 , Val51 , Arg54 and Leu55 , for which their respective alanine
c The Authors Journal compilation c 2010 Biochemical Society
substitution mutants exhibited defective GM-CSF recognition
and/or receptor activation, despite normal levels of cell-surface
expression. These findings clearly implicate the Ig-like domain of
hGM-CSFRα as a crucial determinant of GM-CSF binding and
receptor activation, and highlight the Ig-like domain as a novel
target for the development of therapeutics to ameliorate chronic
inflammatory diseases and multiple sclerosis.
EXPERIMENTAL
Preparation of hGM-CSFRα D1
hGM-CSFRα D1, lacking exons 2 and 3, was constructed by deletion mutagenesis using the QuikChange® method (Stratagene)
with Pfu Turbo DNA polymerase and the following primers:
hGMa del25-113 fwd: cctcctgatcccagagaaatcAGGAAGGGAGGGTACCGC; and hGMa del25-113 rev: GCGGTACCCTCCCTTCCTgatttctctgggatcaggagg (the uppercase and
lowercase letters correspond to the respective 5 and 3 sequences
flanking the deleted region). The deletion of exons 2 and 3 eliminates D1 comprising the residues 25–113 (between the amino
acid sequences IPEK24 and S114 GREG), whilst leaving the signal
peptide encoded by exon 1 intact. A modified form of hGMCSFRα D1 was similarly constructed by insertion mutagenesis
to introduce an N-terminal FLAG epitope to allow better detection
of protein cell-surface expression by flow cytometry.
Site-directed mutagenesis of hGM-CSFRα
Site-directed mutagenesis was carried out using the QuikChange®
method with Pfu Turbo Ultra DNA polymerase. The complete
sequences of mutant GM-CSFRα cDNAs were verified by Big
Dye Terminator cycle sequencing (Applied Biosystems).
Expression constructs
For expression in COS7 cells, the cDNAs encoding hβc and wildtype or mutant GM-CSFRα subunits were subcloned into pCEXV3-Xba, a vector derived from pCEX-V3 [32]. This expression
vector contains the SV40 (simian virus 40) origin of replication,
enhancer and promoter regions, so that, after transfection into
COS7 cells, the plasmid replicates episomally and transcribes
efficiently across the insert. For expression in CTLL2 cells,
cDNAs encoding wild-type or mutant hGM-CSFRα subunits were
subcloned into pEF-IRES-N, and βc were subcloned into pEFIRES-P [33] (a gift from Dr Steve Hobbs, Institute of Cancer
Research, London, U.K.).
Cytokines and radiolabelling
hGM-CSF and mIL-2 (murine IL-2) were produced using the
baculovirus expression system, as described previously [34].
Purified GM-CSF was radiolabelled using the Iodogen reagent,
as described previously for murine IL-3 [24,35]. Radiolabelled
GM-CSF was stored at 4 ◦C and used for up to 10 days. 125 I was
purchased from PerkinElmer Life Sciences and stored at room
temperature (22 ◦C).
Cells and DNA transfections
COS7 cells were maintained in DMEM (Dulbecco’s modified
Eagle’s medium) containing 10 % (v/v) FBS (fetal bovine
serum). CTLL2 cells were maintained in RPMI 1640 containing
10 % (v/v) FBS, 50 μM 2-mercaptoethanol and 10 units/ml
mIL-2. Briefly, COS7 cells were harvested using trypsin and
GM-CSFRα Ig-like domain is critical to receptor activation
309
electroporated with 10 μg of wild-type or mutant GM-CSFRα
either alone or together with 25 μg of wild-type βc at 200 V and
960 μF. Binding studies and antibody staining were performed
on cells 64–68 h after transfection. CTLL2 cell lines stably
expressing wild-type βc and wild-type or mutant GM-CSFRα
subunits were generated in two steps with DNA introduced by
electroporation, essentially as described previously [34].
Equilibrium binding analysis for GM-CSF
‘Hot’ saturation binding experiments were performed on COS7
cells transfected with DNA constructs encoding the relevant
receptors, as described previously [23]. In order to assess the
capacity of hGM-CSFRα subunits to bind hGM-CSF directly,
‘cold’ saturation assays were performed on COS7 cells transfected
with cDNAs encoding wild-type or mutant hGM-CSFRα subunits
in the absence of hβc cDNA. 125 I-Labelled hGM-CSF (1 nM)
was added to 106 cells in 200 μl of binding medium (RPMI
1640 supplemented with 0.5 % BSA and 10 mM Hepes, pH 7.5)
with a serial dilution from a 200-fold excess of unlabelled hGMCSF. Non-specific binding was determined by performing the
assay in the presence of 1 μM unlabelled hGM-CSF. After
incubation for 2.5–3 h at 4 ◦C with intermittent agitation, the
assay was terminated by centrifugation through 2:1 (v/v) dibutyl
phthalate/dinonyl phthalate at 12 000 g for 4 min. The tips of
tubes and visible cell pellets were counted using a Packard 5780
Auto-gamma counter. K d (dissociation constant) values were
determined from specific binding data by using the programs
EBDA and LIGAND [36,37], as described previously [23,24,35].
Flow cytometry
COS7 or CTLL2 cells transfected with cDNAs encoding FLAGtagged hGM-CSFRα D1 were incubated in the presence of a
FITC-conjugated anti-FLAG M2 monoclonal antibody (Sigma)
and then washed before analysis of cell-surface expression was
performed on a FACScan flow cytometer (BD Biosciences). COS7
or CTLL2 cells transfected with cDNAs encoding wild-type or
mutant hGM-CSFRα subunits were incubated in the presence
of purified anti-(human CD116) (10 μg/ml; clone 4H1, mouse
IgG1 ; Biolegend) for 30 min at 4 ◦C. The cells were washed
and incubated with a FITC-conjugated rat anti-(mouse IgG1 )
secondary antibody (5 μg/ml; BD Pharmingen) for 25 min at 4 ◦C
and then washed before analysis of cell-surface expression was
performed on a FACScan flow cytometer (BD Biosciences). For
analysis of hβc expression by FACS, cells were harvested and
washed with ice-cold PBS, and then cells were incubated with
a 1:200 dilution of primary antibody [biotin-conjugated mouse
anti-(human CDw131); 0.5 mg/ml; clone 3D7, cat. no. 554535;
BD Biosciences] in FACS incubation buffer [0.2 % BSA and
5 % (v/v) FBS in ice-cold PBS] for 1 h. After three washes with
FACS washing buffer (0.2 % BSA in ice-cold PBS), the cells
were then incubated with secondary antibody [1:1000 dilution
of streptavadin–PE (phycoerythrin); 0.5 mg/ml; cat. no. 554061;
BD Pharmingen) for 30 min. Cells were washed three times with
FACS washing buffer before being subjected to FACS.
Stably transfected CTLL2 cells expressing wild-type or mutant
hGM-CSFRα subunits and hβc were also sorted on the basis
of hGM-CSFRα expression. To separate two distinct cell
populations, FITC-positive and FITC-negative cells were sorted
using the FACSsorter (BD Bioscience). Dead cells were identified
and eliminated by propidium iodide staining and forward sidescatter gating respectively. Gates for each population were
set so that the two subsets sorted based on FITC staining
Figure 1
Deletion of hGM-CSFRα D1 disrupts GM-CSF binding
(A) Schematic models showing component domains of hGM-CSFRα and hGM-CSFRα D1
(deletion of exons 2 and 3, 280 bp, which corresponds to amino acid residues 25–113,
between IPEK24 and S114 GREG). (B) Scatchard representation of hGM-CSF direct binding
assays, depicting a representative ‘cold’ saturation binding assay performed on COS7 cells
expressing the wild-type hGM-CSFRα alone.
would not overlap when re-analysed. Sorted FITC-positive cells
were immediately suspended in growth medium containing
antibiotics and cultured to obtain sufficient numbers for further
characterization.
Proliferation assays
[3 H]Thymidine incorporation assays were performed, as
described previously [34], to determine the capacity of the wildtype or mutant hGM-CSFRα subunits to deliver a proliferative
signal in CTLL2 stable cell lines co-transfected with hβc in
response to hGM-CSF.
RESULTS
Critical role of hGM-CSFRα D1 in binding GM-CSF
To analyse the functional role of hGM-CSFRα D1 we generated
a truncated version of hGM-CSFRα lacking the first domain
(hGM-CSFRα D1; Figure 1A). To determine the capacity of
this mutant hGM-CSFRα to bind GM-CSF, ‘cold’ saturation
binding assays were performed on transiently transfected COS7
cells using 125 I-labelled human GM-CSF and the K d values were
determined (Table 1). COS7 cells expressing the wild-type GMCSFRα subunit bound 125 I-labelled GM-CSF with low affinity
(Table 1 and Figure 1B), consistent with the original description by
Gearing et al. [38]. In contrast, when we expressed the truncated
GM-CSFRα subunit hGM-CSFRα D1 we were unable to detect
125
I-labelled GM-CSF binding (Table 1). To ensure that the loss of
GM-CSF binding did not arise from loss of receptor expression,
we performed flow cytometry to check that the truncated receptor
c The Authors Journal compilation c 2010 Biochemical Society
310
S. Mirza and others
Table 1 hGM-CSF binding to COS7 cells expressing wild-type or mutant
hGM-CSFRα subunits
Binding was determined using the ‘cold’ saturation binding assay, and the K d values +
− S.E. were
determined from co-analysis of data obtained from separate binding experiments using LIGAND
[37]. −, no binding was detected above the background non-specific binding; n , number of
experiments.
GM-CSFRα
K d (nM)
n
Wild-type
D1
R49A
V50A
V51A
E52A
R54A
L55A
S56A
N57A
N58A
E59A
C60A
S61A
4.8 +
− 0.33
−
12.5 +
− 1.4
7.5 +
− 0.6
−
9.2 +
− 0.6
34.2 +
− 2.6
31 +
− 1.8
7.4 +
− 1.3
5.7 +
− 0.6
12.5 +
− 1.3
7.78 +
−1
−
3.98 +
− 0.3
5
5
1
1
4
1
3
3
1
2
2
2
4
2
Figure 2
was expressed on the cell surface. As it is possible that the anti(GM-CSFRα) antibody epitope is located in D1, we incorporated
a FLAG epitope between the signal peptide and mature hGMCSFRα D1-coding sequence and expressed this variant in COS7
cells. Flow cytometry following anti-FLAG antibody staining
showed that the D1-deleted hGM-CSFRα is expressed on the
cell surface at the same level as the wild-type (see Figure 4).
The bulk of the cells expressed detectable levels of receptor and a
small subset expressed higher levels. The expression data indicate
that deletion of the Ig-like domain does not compromise folding,
trafficking or cell-surface expression. These observations suggest
an obligatory role for the Ig-like domain in low-affinity GM-CSF
binding, analogous to the role of D1 in the hIL-5Rα subunit in
mediating binding to its cognate ligand. In contrast, IL-3Rα D1
contributes to high-affinity binding, but is not obligatory [29,39].
Binding properties of wild-type and mutant hGM-CSFRα in the
presence of hβc
To study the role of GM-CSFRα D1 in the formation of the highaffinity GM-CSF–GM-CSFRα–hβc complex, we co-transfected
COS7 cells with expression vectors encoding hβc and wild-type
Scatchard representation of hGM-CSF ‘hot’ saturation binding assays
Scatchard plots depicting 125 I-labelled GM-CSF ‘hot’ saturation binding data for COS7 cells expressing hβc plus wild-type or mutant GM-CSFRα D1, V51A, R54A, L55A or C60A, as labelled on
the Figure. Data from a representative ‘hot’ saturation binding experiment are shown in each plot with the line-of-best-fit determined by co-analysis of data from several binding experiments using
LIGAND [37]. The derived K d values are shown in Table 2.
c The Authors Journal compilation c 2010 Biochemical Society
GM-CSFRα Ig-like domain is critical to receptor activation
Table 2 hGM-CSF binding to COS7 cells expressing hβc and wild-type or
mutant hGM-CSFRα subunits
Binding was determined using the ‘hot’ saturation binding assay and K d values +
− S.E. were
determined using LIGAND [37]. *To improve the accuracy of determination of the high-affinity
K d , the low-affinity K d was fixed according to the values from Table 1. †High-affinity binding
data sets for L55A gave both one-site and two-site models, consistent with residual though
reduced binding for hGM-CSFRα L55A alone (Table 1). −, a one site binding model was
statistically significant; n , number of experiments.
Kd
hGM-CSFRα
High-affinity (pM)
Low-affinity (nM)
n
Wild-type
D1
V51A
R54A
L55A
65.7 +
− 8.8
481 +
− 39.5
190 +
− 13
211 +
− 8.4
179 +
− 18
148 +
− 7.6†
646 +
− 59.4
4.8*
−
−
−
31*
−
−
7
3
2
2
1
1
3
C60A
or hGM-CSFRα D1 subunits and performed ‘hot’ saturation
binding assays using 125 I-labelled hGM-CSF. These binding
studies were performed using hGM-CSFRα subunits that were not
tagged with an N-terminal FLAG epitope. Cells expressing both
wild-type hGM-CSFRα and hβc exhibited two GM-CSF-binding
sites, as illustrated by the curvilinear Scatchard plot (Figure 2).
The K d values of 66 pM (high affinity) and 4.8 nM (low affinity)
(Table 2) are consistent with those reported previously for the
GM-CSFR system [6,38]. When COS7 cells were co-transfected
with hGM-CSFRα D1 and hβc, we observed a single GM-CSFbinding site of intermediate affinity (481 pM), whereas a lowaffinity site corresponding to GM-CSF binding by GM-CSFRα
alone could not be detected (Table 2 and Figure 2). The presence of
the intermediate-affinity site, rather than a high-affinity GM-CSFbinding site, observed with hGM-CSFRα D1 suggests that,
although neither the hGM-CSFRα D1 mutant nor wild-type
hβc bind GM-CSF detectably alone, their co-expression enables
ligand binding. These data suggest that the binding contributions
of hβc partially compensate for the deficiency in binding of hGMCSFRα D1 to GM-CSF.
Identification of critical residues in hGM-CSFRα D1 for binding to
hGM-CSF
The recent publication of the structure of the IL-13Rα1 complex
(Figure 3D) [26] provided, for the first time, a good model for
the α receptors interacting with their ligands and illustrated a
clear role for the C strand within D1 in mediating binding
to its ligands IL-13 and IL-4. As a previous study identified
key residues in hIL-5Rα D1 involved in IL-5 binding [27], we
used these data to verify that IL-13Rα1 was a good model
for hIL-5Rα and its sister receptor hGM-CSFRα. As shown
in Figures 3(A)–3(D), and Supplementary Figure S1(A) (available at http://www.BiochemJ.org/bj/426/bj4260307add.htm), the
structural alignment of IL-13Rα1 and hIL-5Rα gave a precise
positioning of the key IL-5-binding residues close to the relevant
binding residues identified from the IL-13Rα1–IL-13 crystal
structure [26]. Other expected structural features also aligned
well. The lone cysteine residue in the Ig-like domain of hIL-5Rα
was shown previously to be solvent-exposed by virtue of its
covalent modification by isothiazolone compounds [42]. These
data provide further validation for the suitability of hIL-13Rα1
(human IL-13Rα1) as a template for hIL-5Rα structural
alignment, as this cysteine residue is predicted to lie in the E–F
311
loop of hIL-5Rα D1, a solvent-exposed region of the structure
(Supplementary Figure S1A). The disulfides between strands 2A
and 2B and strands 2D and 2E aligned with conserved disulfides in
IL-13Rα1. Similarly, there was alignment of the WSXWS motif
and its associated RXR motif, both of which are involved in the
Trp-Arg zipper. It was therefore likely that a structural alignment
of GM-CSFRα with IL-13Rα1 would also be informative and
this was carried out (Supplementary Figure S1B). It also showed
conservation of expected structural features. A further guide to the
position of the residues of hGM-CSFRα D1 likely to be involved
in cytokine binding was the conserved exon structures of the
genes, as an exon boundary was close to the critical residues in
IL-13Rα1 and hIL-5Rα (Supplementary Figure S1A). The structural alignment of hGM-CSFRα also showed that the
corresponding exon boundary was aligned with the same region
(Supplementary Figure S1B).
The IL-13Rα1 complex showed contacts between residues
Lys76 to Glu81 including the region of the C strand (Figures 3A and
3B) [26]. From the structural alignment model of GM-CSFRα, we
identified 12 candidate GM-CSF-binding residues in the region
of the predicted C strand of D1 and, subsequently, performed
site-directed mutagenesis to generate individual alanine mutants
(Table 1). 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 [15], since
alanine possesses a small hydrophobic side chain which is
often found in solvent-exposed regions of proteins. Furthermore,
alanine substitution does not disrupt the geometry of the main
chain, and the methyl side chain does not cause electrostatic or
steric interference.
Binding properties of hGM-CSFRα mutants containing mutations in
the region of the putative C strand
Each of the 12 alanine mutants of residues in the region of the
predicted C strand were transiently expressed in COS7 cells and
subjected to the ‘cold’ saturation assay to ascertain the effect
of the mutations on low-affinity hGM-CSF binding. Pro53 was
not mutated in the present study, as this substitution is likely to
result in major structural perturbation of the C–C loop, rendering
interpretation of any loss of binding ambiguous. None of the
GM-CSFRα subunits used in these experiments were tagged with
the FLAG epitope at their N-termini. COS7 cells transfected
with the cDNA encoding wild-type GM-CSFRα were found
to bind GM-CSF with low affinity (K d =4.8 nM), as described
above, and the K d values of R49A, V50A, E52A, S56A, N57A,
N58A, E59A and S61A mutant hGM-CSFRα subunits were not
greatly affected (Table 1). In contrast, the R54A and L55A hGMCSFRα subunits showed a reduction in affinity for hGM-CSF
binding of 7- and 6-fold respectively (Table 1), and complete
abolition of hGM-CSF binding was observed with the V51A and
C60A hGM-CSFRα subunits. These results implicate Val51
and Cys60 as key hGM-CSF-binding determinants and suggest
that Arg54 and Leu55 also play an important additional role in
hGM-CSF binding. To determine whether the residues critical
for direct (low-affinity) hGM-CSF binding were also involved in
high-affinity binding, COS7 cells were transiently co-transfected
with expression vectors encoding wild-type hβc and wild-type or
mutant hGM-CSFRα subunits, and ‘hot’ saturation binding assays
were performed in which increasing amounts of 125 I-labelled
hGM-CSF were added (Table 2 and Figure 2). Interestingly,
all of these mutants were capable of binding hGM-CSF with
intermediate-affinity (K d =148–646 pM compared with wild-type
affinity of 66 pM). Mutation of Cys60 gave the strongest effect,
reducing high-affinity binding by a similar amount to D1 deletion.
c The Authors Journal compilation c 2010 Biochemical Society
312
Figure 3
S. Mirza and others
Structural homology models of hGM-CSFRα and hIL-5Rα
(A) The structural model of the extracellular domain of GM-CSFRα (left) was generated using FUGUE [40] by homology with the IL-13Rα1 crystal structure (from PDB 3BPO). GM-CSF (PDB 1CSG)
and GM-CSFRα were superimposed on to IL-13 and IL-13Rα1 respectively, from PDB 3BPO using the program Coot [41]. GM-CSF is drawn in grey and GM-CSFRα is in red. (B) Expansion of
the boxed region from (A). Side chains of residues from D1 of hGM-CSFRα that were mutated in the present study due to their potential involvement in the interaction with the ligand are drawn
as blue sticks and are labelled. (C) Homology model of the hIL-5Rα ectodomain, based on the crystal structure of IL-13Rα1, generated using FUGUE [40]. The side chains of residues from D1
that are critically involved in IL-5 interactions are drawn as blue sticks and are labelled. (D) The IL-13–IL-13Rα1 complex drawn from PDB 3BPO. The side chains of residues located at the Ig-like
domain of IL-13Rα1 involved in an anti-parallel β-strand interaction with ligand are drawn as blue sticks and are labelled. Cartoon structural models were drawn using PyMOL (DeLano Scientific;
http://www.pymol.org). (E) Multi-species alignment of D1 and a portion of D2 of GM-CSFRα. Conserved cysteine residues are highlighted in green. Potential binding residues around the predicted
C strand of D1 of hGM-CSFRα are highlighted in turquoise, and the residues identified as involved in ligand binding in the present study are highlighted in red text and are reproduced below the
multi-species alignment in red text. The predicted secondary structure features are marked above the sequence; green arrows represent β-strands.
Cell-surface expression of mutant hGM-CSFRα receptors
To verify that the loss of ligand binding with the V51A,
R54A, L55A and C60A mutant hGM-CSFRα receptors did not
result from a perturbation of the receptor translation, folding
or presence on the cell surface, flow cytometry was used
to detect mutant hGM-CSFRα cell-surface expression. COS7
cells transiently expressing wild-type or mutant hGM-CSFRα
subunits were stained with purified anti-(human CD116) (clone
4H1) and a FITC-conjugated secondary antibody and were
examined by indirect flow cytometry (Figure 4). The level of
cell-surface expression of the V51A, R54A, L55A and C60A
mutant hGM-CSFRα was observed to be equivalent to wild-type
hGM-CSFRα expression with only small variations observed
because of differences in transfection efficiency. COS7 cells
c The Authors Journal compilation c 2010 Biochemical Society
transfected with the empty vector, pcEX-V3-Xba, exhibited no
increase in fluorescence intensity above background when stained
with secondary antibody alone (see Figure 4). Additionally,
hGM-CSFRα mutants were capable of participating in highaffinity hGM-CSF-binding complexes (Table 2), providing further
evidence for their presence on the cell surface. However, it was
also important to examine whether the Ig-like domain may play a
critical role in reorientating receptor subunits to initiate activation
of intracellular signalling by the high-affinity receptor complex,
as investigated below.
Critical role of D1 and Cys60 of hGM-CSFRα in receptor activation
Although the formation of a high-affinity complex is a prerequisite
for receptor activation, it is not necessarily sufficient for
GM-CSFRα Ig-like domain is critical to receptor activation
Figure 4
313
Cell-surface expression of wild-type and mutant hGM-CSFRα subunits as detected by flow cytometry
COS7 cells transfected with cDNAs encoding wild-type or mutant GM-CSFRα after 64–68 h were stained with purified anti-(human CD116) antibody and FITC-conjugated secondary antibody
[FITC-conjugated rat anti-(mouse IgG1 ) antibody]. Cells stained with only the FITC-conjugated antibody were used as a control for each assay (depicted by the grey-shaded area). hGM-CSFRα D1
cell-surface expression was examined using a modified form of the receptor bearing an N-terminal FLAG-tag, followed by staining with an anti-FLAG antibody. COS7 cells transfected with empty
vector gave the same results as the control. FACS analyses of wild-type and mutant GM-CSFRα subunit expression in CTLL2 stable cell lines (both unsorted and sorted for GM-CSFRα levels) are
shown in Supplementary Figure S2(B) (at http://www.BiochemJ.org/bj/426/bj4260307add.htm).
signalling. We therefore considered it important to correlate the
binding data with an unequivocal signalling response, in this case
proliferation. hβc DNA was stably transfected into the mIL-2dependent lymphoid cell line CTLL2, via the pEF-IRES-P vector,
which allows for selection by puromycin resistance. The pEFIRES expression vector utilizes the strong EF-1α (elongation
factor-1α) promoter, which is highly active in CTLL2 cells and the
expression of the puromycin-resistance gene is indicative of
the expression of the cloned receptor. Verification of hβc expression was carried out by FACS (Supplementary Figure S2A, available at http://www.BiochemJ.org/bj/426/bj4260307add.htm).
Puromycin-resistant cells were subsequently separately transfected with wild-type hGM-CSFRα or the mutant hGM-CSFRα
subunits (D1, V51A, R54A, L55A or C60A) in the vector pEFIRES-N, which encodes G418 resistance. Antibiotic-resistant
cells were selected in the presence of mIL-2, and proliferation
assays carried out to determine responsiveness to hGM-CSF after
mIL-2 was removed. hGM-CSFRα D1 and C60A exhibited
a greatly reduced response to GM-CSF compared with cells
expressing the wild-type receptor (Figures 5A and 5B). They
not only required markedly higher concentrations of ligand for
growth stimulation, but the maximum stimulation of the cells was
also much lower than that of the wild-type. These mutants were
therefore studied in detail.
Duplicate CTLL2 cell lines expressing wild-type hGMCSFRα, hGM-CSFRα D1 and hGM-CSFRα C60A, together
with hβc, were generated from separate transfections. Following
selection of antibiotic-resistant cells in the presence of mIL-2,
the stable cell lines were sorted by FACS to enrich for high
hGM-CSFRα expression, and both the sorted and unsorted stable
cell lines were compared. Expression of wild-type and mutant
hGM-CSFRα was verified by FACS (Supplementary Figure S2B).
In each case the sorting further enhanced the level of hGM-
CSFRα expression in the cell populations. The hGM-CSFRα
D1 expressed in these experiments possessed an N-terminal
FLAG epitope to allow sorting using an anti-FLAG antibody,
whereas wild-type- and C60A hGM-CSFRα-expressing cells
were sorted using an anti-(hGM-CSFRα) antibody. Continued
high expression of hβc in each of the stable cell lines was also
verified (results not shown).
‘Hot’ saturation binding assays were used to measure highaffinity hGM-CSF binding (Table 3 and Figure 5D). The
unsorted wild-type hGM-CSFRα/wild-type hβc-expressing cells
gave a K d value of 91 pM for high-affinity binding, which
decreased to 50 pM in the FACS-sorted cells. The number
of low-affinity-binding sites (attributable to hGM-CSFRα) was
significantly higher in the sorted cells, as expected, but the
number of high-affinity-binding sites was only slightly increased
(Table 3). In the case of hGM-CSFRα D1, the K d value for
high-affinity binding in unsorted cells was much higher than that
for their wild-type counterparts (314 pM), and there was a 25-fold
reduction in the number of binding sites. This was despite similar
receptor expression as measured by FACS (Supplementary Figure
S2A), suggesting that the mutant hGM-CSFRα complex with hβc
may be unstable. Although the hGM-CSFRα subunit expressed in
this CTLL2 cell line bears an N-terminal FLAG-tag, a comparable
K d value for high-affinity binding (481 pM) was observed in ‘hot’
saturation hGM-CSF-binding assays when hGM-CSFRα D1
lacking a FLAG-tag was co-expressed with wild-type hβc in
COS7 cells (Figure 2 and Table 2). With the sorted hGM-CSFRα
D1/wild-type hβc-expressing cells, the number of high-affinitybinding sites was greatly increased, together with a lowering of
the K d value to 135 pM. This suggests that some stabilization
of the complex with hβc is occurring in the cells expressing
higher levels of hGM-CSFRα D1. hGM-CSFRα C60A/wildtype hβc-expressing cells also showed a much higher K d value
c The Authors Journal compilation c 2010 Biochemical Society
314
Table 3
S. Mirza and others
hGM-CSF binding to CTLL2 cells expressing hβc and wild-type or mutant hGM-CSFRα subunits
*Each stable CTLL2 transfectant population was sorted by FACS for elevated expression of hGM-CSFRα, and the binding characteristics of sorted (+) and unsorted (−) cells were compared.
†Binding was determined using the ‘hot’ saturation binding assay and K d values +
− S.E. were determined using LIGAND [37]. ‡To improve the accuracy of determination of the high-affinity K d , the
low-affinity K d was fixed at 4.8 nM from Table 1. n , number of experiments.
High-affinity
Low-affinity
FACS-sorted (hGM-CSFRα)*
hGM-CSFRα
K d (pM)†
Sites/cell (n )
K d (nM)
Sites/cell (n )
n
−
+
−
+
−
+
Wild-type
Wild-type
D1
D1
C60A
C60A
91 +
− 17.5
50 +
− 20
314 +
− 19
135 +
− 10.6
451 +
− 54
465 +
− 55.8
3035 +
− 315
3613 +
− 568
120 +
− 60
6949 +
− 390
361 +
− 32
1433 +
− 144
4.8‡
4.8‡
−
−
−
−
13610 +
− 1497
34085 +
− 3215
−
−
−
−
2
2
2
2
1
2
Figure 5
Proliferation of CTLL2 cell lines stably expressing wild-type or mutant hGM-CSFRα plus hβc
Plots depict the percentage of maximal growth response to a serial dilution of hGM-CSF for CTLL2 cells stably co-expressing wild-type hβc with: (A) wild-type or D1 GM-CSFRα; (B) wild-type,
V51A, R54A, L55A or C60A GM-CSFRα; and (C) wild-type, D1 or C60A GM-CSFRα using cells sorted by FACS for GM-CSFRα expression. The data shown in (A–C) are representative of triplicate
experiments. (D) Scatchard plots of 125 I-labelled GM-CSF ‘hot’ saturation binding data for CTLL2 cells (unsorted or sorted) expressing hβc plus wild-type, D1 or C60A mutant GM-CSFRα as
labelled in the Figure. Data from a representative ‘hot’ saturation binding experiment are shown in each plot with the line of best fit determined from co-analysis of multiple experiments using LIGAND
[37]. The derived K d values are shown in Table 3.
c The Authors Journal compilation c 2010 Biochemical Society
GM-CSFRα Ig-like domain is critical to receptor activation
for high-affinity binding (451 pM) and an 8-fold reduction in the
number of binding sites in unsorted cells. The number of binding
sites increased 4-fold in the sorted cells, but the K d value for
high-affinity binding did not change (Table 3).
The sorted stable transfectants were also tested in proliferation
assays to assess their responsiveness to hGM-CSF after mIL-2
was removed. Sorting for higher levels of expression of hGMCSFRα D1 improved the growth signalling compared with
unsorted cells, but it was still noticeably abnormal in terms
of both responsiveness and the stimulation plateau that was
reached (Figure 5C). Sorting did not improve the abnormal
growth signalling shown by hGM-CSFRα C60A (Figure 5C).
The abnormal growth signalling shown by cells co-expressing
C60A or D1 hGM-CSFRα and wild-type hβc were similar,
both showing a dramatic reduction in the stimulation plateau
as well as reduced responsiveness. The data indicate defective
growth signalling by the two mutants, and that hGM-CSFRα
D1 is critical for both normal binding and for receptor
activation.
An alignment of extracellular domains of GM-CSFRα from
different species was carried out to examine the conservation of
important residues including the ones highlighted in the present
study. Part of this alignment is shown in Figure 3(E). The cysteine
residues in D2 comprising the two disulfides between strands
A and B and strands D and E were found to be absolutely
conserved. There was no similar evidence of disulfides in D1,
where cysteine residues vary in number and position across
species. The key residue Cys60 was highly conserved and Val51
was conserved as either valine or isoleucine. Arg54 and Leu55 also
some showed conservation between species, with Arg54 conserved
largely as arginine or lysine and Leu55 as leucine, valine or
isoleucine.
DISCUSSION
To date, the definition of ligand-binding interfaces in the cytokine
class I receptor family has focused predominantly on epitopes
formed by CRMs, as typified by the GHR. The contributions
of other domains within cytokine receptors, such as the Ig-like
domain, have until recently remained poorly understood in the
absence of structural information. The recently reported X-ray
crystal structures of IL-4 and IL-13 in complex with the IL-13Rα1
ectodomain have provided key insights into the contribution of IL13Rα1 D1, in combination with its canonical adjacent CRM, in
binding its cytokine ligands [26]. These crystal structures provide
a template for extending our knowledge of how cytokine receptors
with analogous topologies, such as the hβc co-receptors, the IL3Rα, IL-5Rα and GM-CSFRα, may utilize their D1 in ligand
recognition and receptor activation. The importance of the Ig-like
domains of IL-3Rα and IL-5Rα in binding their cognate ligands
is now well-established [27–29,39], but oddly, the role of the
GM-CSFRα D1 has escaped scrutiny. Furthermore, no electron
density was observed in the recently described GM-CSF–GMCSFRα–βc X-ray crystal structure [31] to clarify the role of
GM-CSFRα D1. In light of our recent studies establishing
critical roles for the IL-3Rα subunit Ig-like domain in ligand
binding, receptor activation and downstream signalling to control
haemopoietic cell differentiation [29], we describe in the present
studies the essential role that the related hGM-CSFRα D1 plays in
mediating hGM-CSF binding and governing receptor activation.
Initially, we set out to determine whether GM-CSFRα D1 is
critically required for: (i) directly binding hGM-CSF with low
affinity; (ii) high-affinity binding of hGM-CSF in the presence
of hβc; (iii) receptor activation and the initiation of intracellular
315
signalling pathways; and/or (iv) intracellular trafficking and cellsurface localization, a function attributed previously to D1 of
IL-6Rα [43]. We deleted exons 2 and 3 of hGM-CSFRα, to
eliminate D1 (residues 25–113) without disrupting the signal
peptide encoded by exon 1, expressed the resulting construct in
COS7 cells in the absence or presence of the shared receptor
subunit hβc, and assessed binding to 125 I-labelled hGM-CSF. Our
data clearly demonstrate the necessity for GM-CSFRα D1 for
direct binding to hGM-CSF, since no hGM-CSF binding was
detectable in these experiments, despite confirmation that the
mutant receptor was expressed on the cell surface at wild-type
levels (Table 1 and Figure 4). In the presence of hβc, lowaffinity GM-CSF binding was again not observed, but rather a
single binding site corresponding to reduced hGM-CSF binding
relative to wild-type high-affinity binding (Table 2 and Figure 2).
The existence of a single binding site is illustrated by the
linearity of fit in Scatchard plots for binding experiments when
hGM-CSFRα D1 and hβc were co-expressed, compared with
the curvilinearity of wild-type hGM-CSFRα and hβc Scatchard
plots, characteristic of a two-site fit (Figure 2). Thus additional
binding sites provided by hβc partially compensate for the
inability of the hGM-CSFRα D1 to bind directly to hGMCSF. Similar compensation effects by the presence of hβc have
been reported previously. For example, mutation of Asp112 in
GM-CSF abrogated detectable binding to hGM-CSFRα alone,
yet this mutant exhibited near wild-type activity in cells coexpressing hGM-CSFRα and hβc [30]. Notably, the affinity of
hGM-CSF binding by the hGM-CSFRα D1 and hβc subunits
was reduced compared with the wild-type hGM-CSFRα and hβc
complex, thereby indicating a critical role for the hGM-CSFRα Iglike domain in stabilizing GM-CSF binding within the signalling
complex. To the best of our knowledge, these data represent the
first evidence establishing the importance of the Ig-like domain
of hGM-CSFRα in hGM-CSF binding.
Although COS7 cells co-expressing the hGM-CSFRα D1
and hβc showed medium-affinity binding of hGM-CSF, when
these subunits were stably expressed in the factor-dependent
CTLL2 cell line, a defective growth response was observed. The
observation of hGM-CSF binding by cells co-expressing hGMCSFRα D1 and hβc confirms their cell-surface expression, in
agreement with the FACS analysis, indicating that deletion of D1
did not disrupt the hGM-CSFRα translation, folding or transport
to the membrane. This result emphasizes the importance of GMCSFRα D1 not only in ligand binding, but also in the formation
of an efficient signalling receptor complex, and underscores the
complexity of the receptor system, where high-affinity GM-CSF
binding is a necessary, but not sufficient, step to instigate receptor
activation. The data reported in the present study suggest that
GM-CSF binding by the hGM-CSFRα–hβc complex is necessary
to reorientate the receptor subunits to initiate intracellular signal
transduction and that this process critically relies on the Ig-like
domain of the hGM-CSFRα subunit. The concept that activation
of receptor signalling requires appropriate receptor orientation
was originally established for the EPO receptor [44]. Recent
FRET (fluorescence resonance energy transfer) studies of IL-5
binding to its receptor suggest binding by a pre-existing receptor
hetero-oligomer induces reorientation of receptor ectodomains
and rearrangement of the transmembrane and cytoplasmic regions
to initiate signal transduction [45].
In order to deduce the epitope within hGM-CSFRα D1
that plays a key role in GM-CSF recognition and receptor
activation, we generated a structural model of the hGM-CSFRα
ectodomain by homology with the recently reported IL-13Rα1–
IL-13 complex crystal structure [26]. IL-13Rα1 and IL-13 are
suitable structural templates for hGM-CSFRα and hGM-CSF
c The Authors Journal compilation c 2010 Biochemical Society
316
S. Mirza and others
respectively, as both α chains share the Ig-like–CRM topology in
their ectodomains and both cytokines are short-chain four-helixbundle proteins with an up-up-down-down topology [26,46]. To
test the validity of our approach we initially carried out an
alignment with hIL-5Rα as the critical residues in D1 of this
receptor have been identified. The alignment showed correct
positioning of key structural features as well as the key D1-binding
residues Asp55 and Asp56 within the predicted C strand and the
adjacent Tyr57 (Figure 3C) [27]. Interestingly, the alignment also
indicated that hIL-5Rα may utilize a surface-bound disulfide
in D3 analogous to that in IL-13Rα1, which is involved in
IL-13 binding. From a similar alignment with GM-CSFRα, 12
candidate GM-CSF contact residues were identified in the region
of the predicted hGM-CSFRα D1 C strand (Figures 3A and 3B),
a region established previously to be important in the related
IL-5Rα, as alanine substitution of Asp55 and Asp56 within the
predicted C strand and the adjacent Tyr57 disrupted IL-5 binding
[27]. We examined the contributions of these 12 residues in the
region of the putative hGM-CSFRα D1 C strand to hGM-CSF
binding and receptor activation by characterizing their alanine
mutant analogues. Interestingly, alanine substitution of hGMCSFRα Val51 and Cys60 resulted in the complete loss of detectable
low-affinity GM-CSF binding, and mutation of the neighbouring
Arg54 and Leu55 caused respective 8- and 7-fold reductions in
low-affinity GM-CSF binding. When these mutant hGM-CSFRα
subunits were co-expressed with hβc, the most severe disruption
of high-affinity hGM-CSF binding was observed for C60A
GM-CSFRα, which exhibited a 10-fold lower affinity for hGMCSF, comparable with the effect of D1 deletion. The reduction in
GM-CSF high-affinity binding observed for hGM-CSFRα C60A
was borne out in studies of its capacity to deliver a proliferative
signal when installed into a factor-dependent cell line, CTLL2,
stably expressing wild-type hβc. These cells showed a retarded
growth response in a hGM-CSF titration and a low maximum
stimulation. This growth assay confirmed that Cys60 within hGMCSFRα D1 is a key determinant of both GM-CSF recognition
and receptor activation, whereas analogous CTLL2 cell lines
co-expressing hβc and V51A, R54A and L55A mutant hGMCSFRα subunits exhibited near wild-type proliferative responses
in a hGM-CSF titration. These data indicate that Val51 , Arg54
and Leu55 of hGM-CSFRα D1 individually play minor roles
in facilitating hGM-CSF binding, but do not appear to play
important roles in receptor activation. The role played by the
critical residue Cys60 in ligand binding and subunit reorientation
within the signalling complex is not completely clear. Alignment
of GM-CSF ectodomains from a number of species indicated that
Cys60 is highly conserved (Figure 3E), but gives no indication of
other conserved cysteine residues that could partner it in disulfide
bond formation. Similarly, it does not align with cysteine residues
involved in disulfides in hIL-13Rα1 D1 (Supplementary Figure
S1). Thus no clear evidence was obtained of involvement of
Cys60 in disulfide bond formation. A postulated mechanism for
hGM-CSFR activation involves disulfide cross-linking between
an unidentified cysteine residue in hGM-CSFRα and hβc D1 D–
E loop [47]. It seems unlikely that Cys60 would be involved in such
a cross-link as it plays a critical role in ligand binding. A more
complete understanding of the role of Cys60 awaits elucidation
of an X-ray crystal structure with electron density for the hGMCSFRα D1 ligand interaction.
AUTHOR CONTRIBUTION
Shamaruh Mirza, Jinglong Chen, James Murphy and Ian Young designed the research.
Shamaruh Mirza, Andrew Walker and James Murphy performed the research. Shamaruh
c The Authors Journal compilation c 2010 Biochemical Society
Mirza, Jinglong Chen and Ian Young analysed the data. Shamaruh Mirza, James Murphy
and Ian Young wrote the paper.
ACKNOWLEDGEMENTS
We thank J. Olsen for excellent technical advice; Dr Steve Hobbs for providing the pEFIRES expression vectors; and Tracy Willson (The Walter and Eliza Hall Institute for Medical
Research, Parkville, Australia) for providing the hGM-CSFRα cDNA. We are also grateful
to the John Curtin School of Medical Research Microscopy & Cytometry Facility for
FACS and ACRF Biomolecular Resource Facility, the Australian National University for
automated sequencing of plasmids.
FUNDING
This work was supported by the National Health and Medical Research Council of Australia
(NHMRC) [grant numbers 471481]. J.M.M. is the recipient of a NHMRC CJ Martin
(Biomedical) Fellowship.
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Received 11 November 2009/13 January 2010; accepted 15 January 2010
Published as BJ Immediate Publication 15 January 2010, doi:10.1042/BJ20091745
c The Authors Journal compilation c 2010 Biochemical Society
Biochem. J. (2010) 426, 307–317 (Printed in Great Britain)
doi:10.1042/BJ20091745
SUPPLEMENTARY ONLINE DATA
The Ig-like domain of human GM-CSF receptor α plays a critical role in
cytokine binding and receptor activation
Shamaruh MIRZA*, Andrew WALKER*, Jinglong CHEN*, James M. MURPHY†1 and Ian G. YOUNG*1
*Structural Biology Program, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia, and †Molecular Medicine Division, The Walter
and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia
Figure S1
Structural alignment between hIL-13Rα1, hIL-5Rα and hGM-CSFRα
(A) Structural alignment between hIL-13Rα1 (hs3bpoc) and hIL-5Rα (hil5a) performed by FUGUE [1]. Residues involved in IL-5 binding are in red text. The vertical black lines represent the exon
boundary in the first domain of hIL-13Rα1 and hIL-5Rα close to the C strand. (B) Structural alignment between hIL-13Rα1 (hs3bpoc) and hGM-CSFRα (hgma). The key residues involved in
hGM-CSF binding are in red text. The vertical black lines represent the exon boundary in the first domain of hIL-13Rα1 and hIL-5Rα close to the C strand. The conserved WSXWS motif and RXR
motif are highlighted in purple and the conserved cysteine residues are in green. Residues in blue and orange correspond to β-strand and 310 -helix structure elements respectively. The residues
accessible and inaccessible to solvent are in lowercase and uppercase type respectively. Residues involved in hydrogen bonding to main chain amide and main chain carbonyl are shown in boldface
type and underlined respectively. Residues forming disulfide bonds are indicated by a cedilla.
1
To whom correspondence should be addressed (email [email protected] or [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
S. Mirza and others
Figure S2 Cell-surface expression of hβc and wild-type or mutant hGM-CSFRα subunits as detected by flow cytometry in sorted and unsorted stably
transfected CTLL2 cell lines
(A) CTLL2 cells expressing hβc. These cells were used as the parental cell line for transfection with wild-type or mutant hGM-CSFRα. (B) Stable CTLL2 cell lines expressing wild-type or hGM-CSFRα
D1 or C60A together with hβc. Cell lines were either unsorted or had been previously sorted for elevated hGM-CSFRα expression as indicated in the Figure.
REFERENCE
1 Shi, J., Blundell, T.L. and Mizuguchi, K. (2001) FUGUE: sequence-structure homology
recognition using environment-specific substitution tables and structure-dependent gap
penalties, J. Mol. Biol. 310, 243–257
Received 11 November 2009/13 January 2010; accepted 15 January 2010
Published as BJ Immediate Publication 15 January 2010, doi:10.1042/BJ20091745
c The Authors Journal compilation c 2010 Biochemical Society