Interaction of Granulocyte Colony-stimulating Factor (G-CSF)
with Its Receptor
EVIDENCE THAT Glu19 of G-CSF INTERACTS WITH
Arg288 OF THE RECEPTOR*
Judith E.
Layton
,
Grant
Shimamoto§,
Tim
Osslund§,
Annet
Hammacher¶,
David K.
Smith¶
,
Herbert R.
Treutlein¶, and
Tom
Boone§
From the Ludwig Institute for Cancer Research, Melbourne Tumour
Biology Branch, the ¶ Ludwig Institute for Cancer Research and the
Cooperative Research Centre for Cellular Growth Factors,
Parkville 3050, Australia, and § Amgen Inc.,
Thousand Oaks, California 91320
 |
ABSTRACT |
Granulocyte colony-stimulating factor (G-CSF)
forms a tetrameric complex with its receptor, comprising two G-CSF and
two receptor molecules. The structure of the complex is unknown, and it
is unclear whether there are one or two binding sites on G-CSF and the
receptor. The immunoglobulin-like domain and the cytokine receptor
homologous module of the receptor are involved in G-CSF binding, and
Arg288 in the cytokine receptor homologous module is
particularly important. To identify residues in G-CSF that interact
with Arg288, selected charged residues in G-CSF were
mutated to Ala. To clarify whether there are two binding sites, a
chimeric receptor was created in which the Ig domain was replaced with
that of the related receptor gp130. This chimera bound G-CSF but could
not transduce a signal, consistent with failure of dimerization and
loss of one binding site. The G-CSF mutants had reduced mitogenic
activity on cells expressing wild-type receptor. When tested with the
chimeric receptor, all G-CSF mutants except one (E46A) showed reduced
binding, suggesting that Glu46 is important for interaction
with the Ig domain. On cells expressing R288A receptor, all the G-CSF
mutants except E19A showed reduced mitogenic activity, indicating that
Glu19 of G-CSF interacts with Arg288 of the receptor.
 |
INTRODUCTION |
Granulocyte colony-stimulating factor
(G-CSF)1 is a member of a
family of cytokines that have a four-
-helical bundle structure, with
the four helices conventionally labeled A-D from the N terminus (1).
The structures of human, bovine, and canine G-CSF have been determined
by x-ray crystallography (2, 3) and NMR spectroscopy (4, 5). In
addition to the four main
helices, there is a short 310
helix in the A-B loop. The main biological activities of G-CSF are the
proliferation, differentiation, and survival of cells of the neutrophil
lineage (6, 7). These responses are initiated by interaction with a
specific receptor (G-CSF-R) which is expressed on neutrophils, their
precursors, and some leukemic cell lines (6, 7). Binding of G-CSF
causes receptor dimerization and activation of signaling cascades such as the Jak-STAT and mitogen-activated protein kinase pathways (8).
The receptor extracellular region comprises six structural domains as
follows: an N-terminal immunoglobulin-like (Ig) domain, followed by a
cytokine receptor homologous module (CRHM) (containing two fibronectin
type III-like (FNIII) domains as follows: an N-terminal "BN" and a
C-terminal "BC" domain) and three further FNIII domains (9, 10).
This domain structure is closely related to that of gp130, the shared
signal transducer of the IL-6 family of cytokines (11), with which the
G-CSF-R shares 46% sequence similarity in the extracellular region
(12). The CRHM and Ig domain of the G-CSF-R have been implicated in
ligand binding by deletion analysis (10) and mapping of neutralizing
mAbs (13), whereas the three membrane-proximal FNIII domains are not
required for ligand binding but may be important for receptor stability
and/or signal transduction (10, 14). Studies of a soluble form of the
extracellular region of the receptor show that a 2:2 complex of ligand
and receptor form in solution (15), but the structure of the complex is
currently unknown. The BN and BC domains of the G-CSF-R have been
expressed individually, and each has been shown to bind G-CSF with low
affinity (about 10
7 M) in a 1:1 complex (16,
17). The structure of BC has been determined recently by NMR
spectroscopy, and the data indicated that the F-G loop of this domain
is likely to be involved in ligand recognition (18). However, it is not
yet clear whether there are one or two ligand-binding sites on the
G-CSF-R. Horan et al. (14, 15) concluded that there is
probably one ligand-binding site with dimerization caused by
receptor-receptor interaction. On the other hand, studies on the
complexes formed with G-CSF and two soluble receptor fragments (Ig-BN
and the CRHM) indicated that each G-CSF-R in the complex has two
binding sites for G-CSF, one in the CRHM and one requiring the Ig
domain (19). In addition, this study established that the Ig domain of
the G-CSF-R is required for receptor dimerization (19). Similarly, the
Ig domain of gp130 is required for receptor complex formation and
signaling by interleukin-6 (IL-6) (12).
We recently identified several residues in the CRHM that interact with
G-CSF (20), forming a binding site similar to the growth hormone
receptor-binding site (21). Of these residues, Arg288 in
the F-G loop of the BC domain appeared to be the most important for
G-CSF binding, in agreement with the study of Yamasaki et al. (18). This large, positively charged residue might interact with a negatively charged Glu or Asp residue in G-CSF. Mutagenesis studies of G-CSF have identified several charged residues that are
important for receptor binding, including Glu19,
Lys23, Glu46, and Asp112 (22, 23).
In both these studies, Glu19 in the A helix of G-CSF was
identified as particularly important and appeared to be a possible
candidate for interaction with Arg288. In the present
study, to identify which G-CSF residues interact with
Arg288 in the receptor, we have mutated Glu19,
Lys23, Glu46, and Asp112 and
compared the activity of these G-CSF mutants on cells expressing wild-type (WT) and mutant (R288A) receptors. In addition, to clarify further whether there are one or two binding sites on G-CSF, a chimeric
receptor was constructed in which the Ig domain of gp130 replaced the
G-CSF-R Ig domain. This chimera was used to determine whether the above
G-CSF residues interacted with the CRHM-binding site or the putative Ig
domain-binding site.
 |
EXPERIMENTAL PROCEDURES |
Preparation of G-CSF Mutants--
Human G-CSF mutants were
prepared by oligonucleotide-directed mutagenesis (24), and the
mutations were confirmed by sequencing the complete G-CSF cDNA. The
G-CSF mutant proteins were expressed in Escherichia coli and
purified essentially as described previously (25). Samples of mutant
and WT rhG-CSF were analyzed by reverse phase high pressure liquid
chromatography to confirm homogeneity.
G-CSF-R Mutants and Chimeric Receptor--
The human G-CSF-R
mutants were prepared as described previously (20). To generate the Ig
domain chimeric receptor (gp130-Ig)GR, EcoRV restriction
enzyme sites were introduced in both the G-CSF-R and gp130 between the
Ig and CRH domains as described for the G-CSF-R (12, 13)
(oligonucleotide for gp130,
5'-GGAATCACAATAATTAGCGGATATCCTCCAGAAAAA-3'). A
BstXI-EcoRV fragment of gp130 was ligated with an
EcoRV-XbaI fragment of G-CSF-R into
BstXI- and EcoRV-digested pcDNA1Amp
(Invitrogen, The Netherlands). The chimeric receptor was excised with
HindIII and XbaI and ligated with linkers that
contained XbaI and HindIII sites. The receptor
was then subcloned into the XbaI site of pEFBOS expression
vector (26). The chimera encoded residues
Met1-Gly123 of gp130 followed by residues
Tyr98-Phe813 of the G-CSF-R (residues numbered
as in Ref. 27). The receptors were transfected into the murine pro-B
cell line, Ba/F3 (28), as described previously (20).
Flow Cytometry--
Analysis of receptor expression by flow
cytometry was performed as described previously (13) using the
following mAbs: LMM741, which binds to the G-CSF-R FNIII domains (13),
LMM775, which binds to the G-CSF-R Ig domain (13), and GPZ35, which
binds to gp130 (29).
G-CSF Binding Assay--
Tyr1,3-hG-CSF (provided by Kirin,
Japan) was iodinated using IODO-GEN (Pierce) as described previously
(13). The ability of each batch to bind was at least 60%, and the
specific activity was 4-10 × 107 cpm/µg as
determined by self-displacement analysis (30). Binding affinity of
G-CSF mutants was determined by titration of mutants in the presence of
125I-Tyr1,3-G-CSF (~100 pM for WT-GR and 200 pM for (R288A)GR, (R288E)GR, and (gp130-Ig)GR). Data were
analyzed with the "drug" section of the LIGAND program (31)
(Biosoft, Cambridge, UK).
G-CSF Proliferation Assay--
Proliferation of Ba/F3 cells
expressing WT and mutant G-CSF-R was determined by measuring
incorporation of [methyl-3H]thymidine (ICN
Pharmaceuticals, Irvine, CA) as described previously (20).
 |
RESULTS |
Preparation and Characterization of (gp130-Ig)GR--
Because we
believed that the evidence for two separate ligand-binding sites in the
G-CSF-R was stronger than the evidence for a single site, we first
wanted to determine which of the G-CSF residues Glu19,
Lys23, Glu46, or Asp112 were
required for binding to each site. Initially we deleted the Ig domain,
to leave only the CRHM site, but found that this receptor was poorly
expressed and apparently not folded correctly as determined by mAb
binding (13) (data not shown). Therefore a chimeric receptor containing
the Ig domain of the related receptor chain gp130 ((gp130-Ig)GR) was
constructed and expressed in Ba/F3 cells. This construct expressed well
and was tested for binding of anti-gp130 and anti-G-CSF-R mAbs (Fig.
1A). As expected, cells expressing (gp130-Ig)GR bound the mAb against the G-CSF-R FNIII domains
(LMM741) but not the mAb against the G-CSF-R Ig domain (LMM775). The
recognition site of GPZ35 on gp130 was not previously known. Staining
of (gp130-Ig)GR with this mAb establishes that it recognizes the Ig
domain. The chimera bound WT G-CSF with reduced affinity (see below)
but had no detectable activity in a proliferation assay (Fig.
1B), suggesting that the receptor cannot dimerize correctly.

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Fig. 1.
Characterization of the chimeric (gp130-Ig)GR
receptor. A, flow cytometric analysis of mAb binding to
Ba/F3 cells expressing either the wild-type G-CSF-R (WT-GR) or the
chimeric receptor (gp130-Ig)GR. The open histograms
represent the binding of isotype-matched negative control antibodies,
and the filled histograms show the binding of the mAbs
indicated above the panels. B, proliferation of
Ba/F3 cells expressing the WT-GR or (gp130-Ig)GR in response to G-CSF.
Proliferation was measured by determining uptake of
[methyl-3H]thymidine after 48 h
incubation.
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Binding of G-CSF Mutants to the CRHM Site--
To determine which
of the mutated amino acid residues of G-CSF interacted with the CRHM
site, binding of mutant G-CSFs to (gp130-Ig)GR was determined by cold
competition with radiolabeled WT G-CSF (Fig.
2). (E46A)G-CSF bound to (gp130-Ig)GR
with a similar affinity (Kd = 0.50 nM)
to WT G-CSF (Kd = 0.42 nM), indicating
that this residue was not required for binding to the CRHM site and
probably interacts with the Ig domain. No competition was detected with
(E19A)G-CSF and only very weak inhibition with (K23A)G-CSF
(Kd = 11 nM) and (D112A)G-CSF
(Kd = 72 nM), showing that these
residues were required for interaction with (gp130-Ig)GR, presumably
with the CRHM site. Binding of WT G-CSF to the complementary chimera
(GR-Ig)gp130 (12) was not detected (data not shown).

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Fig. 2.
Binding of G-CSF mutants to Ba/F3 cells
expressing the receptor chimera (gp130-Ig)GR. Unlabeled mutant or
WT G-CSF was titrated in the presence of about 200 pM
125I-Tyr1,3-G-CSF and allowed to bind for 4 h at
4 °C. Binding was calculated as a percentage of binding in the
absence of inhibitor after subtraction of nonspecific binding.
Kd values for the mutant G-CSFs were determined by
analysis of the data with the LIGAND program (31) and are mean values
of two independent experiments.
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Proliferation Response of (R288A)GR to the Mutant G-CSFs--
To
determine whether any of the G-CSF residues selected for mutation
interacted with Arg288 in the CRHM of the receptor, we
compared the proliferation of Ba/F3 cells expressing the WT-GR with
cells expressing (R288A)GR in response to the mutant G-CSFs. Typical
results are shown in Fig. 3, and the mean
EC50 values from two independent experiments are summarized
in Table I. The mutant G-CSFs have been
compared with WT G-CSF by calculating the mutant/WT EC50
ratios (Table I). In the case of the WT-GR, the mutations resulted in a
reduced proliferation response of 10-70-fold, with the exception of
(D112A)G-CSF. As shown by the ranges given by the data, the two
experiments gave somewhat different EC50 values; however,
the mutant/WT G-CSF ratios from each experiment were very similar
(range <10%, excluding D112A which was 50%). With (R288A)GR, we were
unable to add sufficient mutant G-CSF to reach plateau responses in
some cases, because of the large effects of some mutant combinations;
thus some EC50 estimates are approximate. Nevertheless, it
is clear that (E19A)G-CSF gave a similar response to WT G-CSF in cells
expressing (R288A)GR, whereas the other G-CSF mutants gave a
substantially reduced response. The effect of combining each of the
K23A, E46A, and D112A G-CSF mutations with the (R288A)GR mutation was
greater than would be predicted from the individual effects of the
G-CSF and G-CSF-R mutations. If the G-CSF and receptor mutations
behaved independently, the mutant/WT ratios (Table I) should have been
similar for the WT and R288A receptors.

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Fig. 3.
Proliferation of Ba/F3 cells expressing WT-GR
or (R288A)GR in response to G-CSF mutants. Uptake of
[methyl-3H]thymidine was measured after
48 h culture.
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To determine whether any of the G-CSF mutations affected the stability
of the G-CSF mutants in culture, recovery of WT G-CSF and the mutants
from the culture supernatant after 48 h incubation was determined
by ELISA (modified from Ref. 32). Recovery of WT G-CSF was 40% and
recoveries of (E19A)G-CSF and (D112A)G-CSF were greater than WT (Table
I). Recovery of (K23A)G-CSF was 7.5% and (E46A)G-CSF was 15%;
therefore, only a small fraction of the reduced activity of these
mutants could be explained by reduced stability. Binding of the mAb
used in the ELISA was not affected by the G-CSF mutations (data not shown).
Binding of G-CSF Mutants to (R288A)GR--
To determine whether
the effects of the mutations on proliferation could be explained by
changes in binding affinity, binding of the G-CSF mutants to Ba/F3
cells expressing WT-GR or (R288A)GR was compared. Typical inhibition
curves for the binding of 125I-Tyr1,3-G-CSF in the presence
of increasing concentrations of mutant or WT G-CSF are shown in Fig.
4. The mean Kd values from two independent experiments with each receptor and G-CSF combination is given in Table II. Fig.
4A shows that the G-CSF mutations had only slight effects on
the affinity of binding to the WT-GR. The K23A, E46A, and D112A G-CSFs
bound less well to (R288A)GR than did WT G-CSF, showing that these
G-CSF residues interacted with residues other than Arg288
on the receptor CRHM or Ig domain (Fig. 4B). In contrast,
(E19A)G-CSF bound as well as WT G-CSF to (R288A)GR, indicating that
Glu19 interacts with Arg288 of the receptor. As
observed with the proliferation data (Table I), the mutant/WT ratios of
the binding affinity of K23A, E46A, and D112A G-CSFs to (R288A)GR were
greater than the corresponding ratios with WT-GR, suggesting a
synergistic effect of receptor and G-CSF mutations.

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Fig. 4.
Binding of G-CSF mutants to Ba/F3 cells
expressing WT-GR or (R288A)GR. Method as in legend for Fig. 2.
125I-Tyr1,3-G-CSF was used at about 100 pM for
WT-GR and about 200 pM for (R288A)GR.
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The Effect of Charge Reversal Mutations--
If the interaction of
Glu19 of G-CSF with Arg288 of the receptor were
predominantly electrostatic, we would predict that charge reversal
mutations would have a greater effect than charge-to-alanine mutations
and that simultaneous charge reversal of both residues might restore
the interaction. The proliferation of Ba/F3 cells expressing WT-GR,
(R288A)GR, or (R288E)GR in response to WT, E19A, and E19R G-CSFs is
shown in Fig. 5 and Table III. With
WT-GR, (E19R)G-CSF had a profoundly reduced activity compared with
(E19A)G-CSF (2000-fold less active), presumably due to charge repulsion
between the two Arg residues. Recovery of (E19R)G-CSF from culture
supernatant was 70%, as measured by ELISA; thus reduced stability did
not explain the low activity. However, with (R288A)GR, this effect disappeared and all three G-CSFs behaved similarly, as would be expected when the charge interaction was removed. This result also
shows that the E19R mutation did not have a detectable effect on the
folding of the mutant G-CSF. The response of (R288E)GR to WT G-CSF was
only slightly reduced in comparison with the response of (R288A)GR,
suggesting that the expected charge repulsion effect was only slight.
However, neither removal of the G-CSF charge in (E19A)G-CSF nor charge
reversal in (E19R)G-CSF improved the response of (R288E)GR. The binding
affinities of the various combinations were also determined and
compared with the proliferation responses (Table
III). The effects on binding affinities
were substantially less than the effects on proliferation, as we have
observed previously (20). This was particularly marked for (E19R)G-CSF
with WT-GR.

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Fig. 5.
Proliferation of Ba/F3 cells expressing
WT-GR, (R288A)GR, or (R288E)GR in response to Glu19 G-CSF
mutants. Uptake of [methyl-3H]thymidine
was measured after 48 h culture.
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Table III
Proliferation and binding affinity of charge reversal mutants on Ba/F3
cells expressing G-CSF-R constructs
Mean ± range of two assays using one cell line expressing each
receptor, including data from Fig. 5.
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|
 |
DISCUSSION |
In the absence of any structural data about G-CSF in complex with
its receptor, we sought to identify the region of G-CSF that interacted
with a previously described receptor-binding site in the CRHM (20), by
using G-CSF mutants. We also investigated whether the Ig domain of the
G-CSF-R contains a binding site for G-CSF. Our data show that
Glu19, Lys23, and Asp112 in G-CSF
interact with the CRHM, whereas Glu46 appears to interact
with the Ig domain of the G-CSF-R (shown schematically in Fig.
6). We conclude that G-CSF has two
distinct receptor-binding sites and that the G-CSF-R has two binding
sites for G-CSF.

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Fig. 6.
Model of the G-CSF receptor-ligand
complex. G-CSF interacts with the CRHM of one receptor (site
II) and the Ig domain of the second receptor (site
III). Interacting amino acid residues are shown.
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With respect to the CRHM-binding site, the G-CSF residues
Glu19 and Lys23 in the A helix and
Asp112 in the C helix form part of a charged binding patch
defined by mutagenesis (22, 23) that is equivalent to growth hormone site II (21). Of the residues interacting with the CRHM, we conclude
that only Glu19 in the A helix interacts with
Arg288 of the receptor. The large loss of activity of
(E19R)G-CSF in comparison with (E19A)G-CSF shows that Glu19
probably makes an electrostatic interaction with Arg288.
Glu19 is sandwiched between Lys16 and
Lys23 in the A helix (2), creating a net single positive
charge in this region that the charge reversal (E19R) converts to a
triple positive charge. This large change in charge could explain the substantial loss of activity of (E19R)G-CSF with the WT-GR. It is
interesting that the binding affinity for this combination was not
proportionately reduced (Table III), suggesting that the mechanism
responsible for the loss of activity in this case is different from
that with (R288A)GR, which had a similar binding affinity but not as
great a loss of activity with any of the G-CSF mutants. The charge
reversal mutation of the receptor, (R288E)GR, did not have as great an
effect as the charge reversal mutation of G-CSF. It seems likely that
the receptor has folded slightly differently to accommodate the
substitution, thus reducing its effect. Arg288 is in the
F-G loop of the BC domain, which was observed to be highly flexible in
an NMR study (18). In addition, Glu is a smaller residue than Arg, thus
rearrangement of the F-G loop may be possible when two Glu residues
are involved in the complex (WT-G-CSF and (R288E)GR) but not when there
are two Arg residues ((E19R)G-CSF and WT-GR).
Similar electrostatic interactions have been described in the human
GM-CSF-receptor complex (33, 34) and the IL-4·IL-4 receptor
chain
complex (35). Arg280 in the
-chain of the GM-CSF-R makes
an electrostatic interaction with Asp112 in the D helix of
GM-CSF (33), whereas Glu21 in the A helix probably
interacts with Tyr365, His367, and
Ile368 of the
-common receptor chain (34). IL-4 binding
to the IL-4 receptor
chain involves a patch of charged residues on
the A and C helices that is likely to interact with charged residues on
the receptor (35). In other cases such as the growth hormone-receptor complex, the most important interactions are hydrophobic (36).
The binding of (E46A)G-CSF and (D112A)G-CSF to (R288A)GR revealed
synergy or cooperativity between the receptor and G-CSF mutations,
because the combined effect was substantially greater than predicted
from the effect of each individual mutation. This observation suggests
that a single mutation may be partially compensated for by the
neighboring residues making alternative contacts in the receptor-ligand
complex. However, when there are two mutations, compensatory contacts
may no longer be possible, leading to greater conformational
perturbation in the complex than was seen with single mutations.
The two recently reported mutagenesis studies of G-CSF differ in their
conclusions about whether there are one or two receptor-binding sites
(22, 23). Both agree that there is a binding site involving charged
residues on the A and C helices of G-CSF, and our data on the effects
of mutating Glu19 and Lys23 in helix A and
Asp112 in helix C further confirm this. Reidhaar-Olson
et al. (22) proposed a second binding site in G-CSF
involving Lys40, Phe144, Val48, and
Leu49. These residues, other than Lys40, are
hydrophobic, suggesting that this site may interact with a hydrophobic
region of the receptor. Like Lys40, Val48, and
Leu49, Glu46 is in the 310 helix in
the A-B loop of G-CSF (2) and may thus form part of this second binding
site. Although Young et al. (23) found no evidence that
Lys40 was important, they did not test the other residues.
We also have found no biological effect of a K40A mutation (data not
shown), but the effects of (E46A)G-CSF provide support for the
existence of a second binding site involving the 310 helix
of G-CSF and the Ig domain of the receptor (site III in Fig.
6). Alternatively, the residues in site III may not interact directly
with the receptor but may be important structurally for stabilizing the
complex with the G-CSF-R.
The chimeric receptor (gp130-Ig)GR was not able to transduce a
proliferative signal in response to G-CSF, although it appeared to be
folded correctly and bound G-CSF, albeit with reduced affinity. The
reduction in binding affinity in itself did not account for the lack of
response because (R288A)GR had a similarly reduced binding affinity and
was able to transduce a response. We conclude that exchange of the Ig
domain has removed a ligand-binding site, creating a receptor that
cannot dimerize and contains a single ligand-binding site in the CRHM.
This is consistent with previous studies of soluble G-CSF-R domains
showing that the Ig domain is necessary for receptor dimerization (19).
In addition, our data on (E46A)G-CSF suggest that the Ig domain
interacts specifically with site III on G-CSF, rather than with the Ig
domain of the second G-CSF-R, to cause dimerization (Fig. 6). A recent
study using Fourier-transformed infrared spectroscopy detected a
conformational change in G-CSF following receptor binding which likely
reflects change of the 310 helix to an
-helix,
consistent with the suggestion that this region is important in the
G-CSF·G-CSF-R complex (37).
Our proposed G-CSF-R complex has many similarities with the complex
proposed for IL-6 with the IL-6 receptor (IL-6R) and gp130 (38). In
this hexameric complex (consisting of two molecules each of IL-6,
IL-6R, and gp130), IL-6 has three sites that are important for complex
formation as follows: site I that binds to the IL-6R (39) and sites II
and III that contact gp130 (40, 41). Site II comprises residues in
helices A and C, and site III includes residues at the beginning of the
D helix and residues in the AB loop of IL-6. G-CSF has a binding site
equivalent to site II in IL-6 (22, 23) and may have a site equivalent
to site III (this study and Ref. 22). Thus the topology of the G-CSF·G-CSF-R complex may resemble that of the IL-6·IL-6R·gp130 complex, without the IL-6R and the interaction with site I. In both
cases, the Ig domain is required for formation of a signaling complex
(10, 12).
We propose that G-CSF first binds through site II to the CRHM of one
receptor. This binding induces a conformational change in G-CSF and the
G-CSF-R that allows interaction of the Ig domain of the second receptor
with the 310 helix region and possibly the N terminus of
the D helix of G-CSF (site III), resulting in receptor dimerization and
formation of a 2:2 (G-CSF·G-CSF-R) complex. There is evidence from
other studies that both G-CSF and the G-CSF-R undergo conformational
changes in the receptor complex (15, 37). Moreover, we have not been
able to detect binding of G-CSF to the Ig domain in a chimeric
receptor, (GR-Ig)gp130 (12), which is consistent with the proposed
requirement for a conformational change in G-CSF and/or the G-CSF-R for
binding to occur. In addition, it suggests that the CRHM is the main
ligand-binding site. There is some evidence that the growth hormone
receptor also undergoes conformational changes after ligand binding
that are required for biological responsiveness (42, 43). Thus,
receptor activation by cytokines may require more than simply achieving
physical proximity of signal transducing receptor components. Our model
of G-CSF·G-CSF-R complex formation accounts for the published data
pertaining to the structure of this complex (15, 19, 20, 37) and
suggests that mutagenesis of the Ig domain of the G-CSF-R will be informative.
 |
ACKNOWLEDGEMENTS |
We thank D. McPhee and F. Connell for expert
technical assistance and L. Souza (Amgen) for providing G-CSF; S. Shimosaka (Kirin, Japan) for providing Tyr-1,3-G-CSF; and S. Nagata
(Osaka University Medical School, Japan) for providing human G-CSF-R cDNA.
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FOOTNOTES |
*
This work was supported by Grants 981130 (to A. H.) and
981133 (to J. L.) from 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.
To whom correspondence should be addressed: Ludwig
Institute for Cancer Research, P. O. Box 2008, Royal Melbourne
Hospital, Parkville, Victoria 3050, Australia. Tel.: 61-3-9341-3155;
Fax: 61-3-9341-3104; E-mail: Judy.Layton{at}ludwig.edu.au.
Current address: Dept. of Biochemistry, University of Hong
Kong, 5 Sassoon Rd., Pok Fu Lam, Hong Kong.
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ABBREVIATIONS |
The abbreviations used are:
CRHM, cytokine
receptor homologous module (containing an N-terminal (BN) and a
C-terminal (BC) domain);
FNIII, fibronectin type III-like;
G-CSF, granulocyte colony-stimulating factor;
G-CSF-R, G-CSF receptor;
GM-CSF, granulocyte-macrophage CSF;
WT, wild type;
IL, interleukin;
(R288A)GR, mutant G-CSF-R in which Arg288 is replaced by Ala;
WT-GR, wild-type G-CSF-R;
gp, glycoprotein;
ELISA, enzyme-linked immunosorbent
assay;
mAb, monoclonal antibody.
 |
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