(Received for publication, June 1, 1995; and in revised form, August 31, 1995)
From the
The carboxyl-terminal domain (BC domain, roughly 100 amino acid
residues) of the cytokine receptor homologous region in the receptor
for murine granulocyte colony-stimulating factor was secreted as a
maltose binding protein fusion into the Escherichia coli periplasm. The murine BC domain was prepared from the fusion
protein by restriction protease factor Xa digestion and was purified to
homogeneity. The purified BC domain specifically and stoichiometrically
bound granulocyte colony-stimulating factor. This result indicates that
the BC domain is also critical for ligand binding, as shown for the
amino-terminal domain of the cytokine receptor homologous region
(Hiraoka, O., Anaguchi, H., Yamasaki, K., Fukunaga, R., Nagata, S., and
Ota, Y.(1994) J. Biol. Chem. 269, 22412-22419). The
tertiary folding and the -sheet structure of the BC domain were
confirmed by NMR spectroscopy. The disulfide bond pattern suggested
from peptide mapping was Cys
-Cys
and
Cys
-Cys
. Disruption of the disulfide
bonds suggested that both bonds are critical for maintaining the
folding of the BC domain, although a BC domain lacking the second bond
still retained ligand binding activity. Mutational analysis of the
WSXWS sequence conserved in the cytokine receptor family
suggested that this motif is critical for protein folding rather than
for ligand binding.
Granulocyte colony-stimulating factor (G-CSF) ()is a
cytokine that plays an essential role in maintaining the number of
neutrophilic granulocytes in peripheral blood and that is responsible
for granulocytosis during inflammation (Nagata, 1990; Nicola, 1989;
Demetri and Griffin, 1991). Since the administration of G-CSF causes
granulopoiesis, it is used to treat patients suffering from
granulopoenia (Lieschke and Burgess, 1992a, 1992b). Investigations of
the ability of ligand binding to the G-CSF receptor to mediate a
specific signal inside the cell may promote clinical applications of
G-CSF. The cDNAs for the murine and human G-CSF receptors (
800
amino acid residues) have been isolated (Fukunaga et al.,
1990a, 1990b). The extracellular region of the G-CSF receptor (
600
amino acid residues) has a composite structure containing an
immunoglobulin-like domain, a cytokine receptor homologous (CRH)
region, and three fibronectin type III domains (Fukunaga et
al., 1990a, 1990b). Among them, the CRH region (
200 amino
acid residues) was predicted to be the ligand binding region (Bazan,
1990). Thus, knowledge of the CRH region is crucial to understanding
the binding mechanism of the G-CSF receptor.
The CRH region was
predicted to be cysteine-rich -structure, which was defined by its
striking homology to receptors for various cytokines, such as
interleukins 2-7, erythropoietin, growth hormone (GH), and
prolactin (Bazan, 1990). The CRH region shares two distinctive features
in the extracellular region: four conserved cysteine residues, known as
the ``cysteine motif,'' are located in the amino-terminal
domain of the CRH region (BN domain), and its carboxyl-terminal domain
(BC domain) contains a ``WSXWS'' motif (Bazan,
1990). It is very likely that the cysteine motif constitutes a domain
for the interaction of the receptor with its cognate ligand (Ullrich
and Schlessinger, 1990). Deletion analysis of the G-CSF receptor cDNA
indicated that the deletion of the BN domain completely abolished
ligand binding (Fukunaga et al., 1991). Recently, Hiraoka et al.(1994), using an Escherichia coli maltose
binding protein (MBP, malE gene product) fusion system, showed
that the BN domain of the G-CSF receptor was a discrete folding unit
that could bind ligand. On the other hand, the G-CSF receptor with a
deletion of the BC domain still retained the ligand binding activity,
although the K
value was increased as
much as 50-fold, as compared with the intact molecule (Fukunaga et
al., 1991). It was reported that the WSXWS motifs of the
interleukin 2 (Miyazaki et al., 1991) and erythropoietin
(Yoshimura et al., 1992) receptors were essential for ligand
binding and signal transduction. These data indicated that the function
of the G-CSF receptor BC domain in ligand binding is still unclear and
suggested that the expression and purification of the BC domain in
quantities suitable for biochemical and structural analyses would be
essential for understanding the ligand binding mechanism of this
receptor.
In the present study, we expressed the BC domain of the G-CSF receptor, using the E. coli MBP fusion system, and purified the product. The purified BC domain specifically bound ligand. Furthermore, we characterized the functions of the WSXWS motif and the cysteine residues.
Figure 1: Schematic representation of the expression and isolation of the mBC domain of the G-CSF receptor. The term Ig-like in the mG-CSF receptor indicates the immunoglobulin-like domain. Fn3 indicates the fibronectin type III region, and TM indicates the transmembrane region. The thin bars in the mBN domain represent conserved cysteine residues. The thick bar in the BC domain represents the WSXWS motif. Numbers correspond to the amino acid numbering of the mG-CSF receptor (Fukunaga et al., 1990a).
The plasmids bearing the two mBC mutants (C224A/C271A and
C242A/C285A) were modified by substituting the codons for each pair of
cysteine residues (Cys,Cys
and
Cys
,Cys
) with alanine codons (TGC
GCT,
TGC
GCT; TGT
GCT, TGC
GCT), respectively. Plasmids for
the four mBC mutants (W294A, S295A, W297A, S298A) were constructed by
changing the codons for the cysteine residues (Trp
,
Ser
, Trp
, Ser
) to serine
codons (TGG
GCT, AGC
GCT, TGG
GCT, AGC
GCT),
respectively.
Figure 2: Purification of the mBC domain. Non-reducing 0.1% SDS, 15% PAGE analysis of proteins present in various stages of the purification is shown. The gel was stained with Coomassie Brilliant Blue. Lane 1, the eluate from the butyl-Toyopearl column; lane 2, the eluate from the S-Sepharose column; lane 3, the eluate from the Q-Sepharose column (before factor Xa digestion); lane 4, the factor Xa digest; lane 5, the eluate from the S-Sepharose column. Lane M shows the molecular weight standards. The numbers show the sizes (kDa) of the marker proteins. The faint band above the main 12.5-kDa band in lanes 4 and 5 disappeared in the electrophoresis under reducing conditions. This faint band is also not detected in the protein of the second disulfide bond mutant C242A/C285A by SDS-PAGE, even under non-reducing conditions. Thus, this band should be derived from a protein that formed an unexpected disulfide bond during the electrophoresis.
Figure 3:
NMR of the mBC domain. The H-NMR spectrum was measured on a 500-MHz spectrometer
(Bruker AM-500) at 20 °C. The purified mBC domain was dissolved in
20 mM sodium phosphate buffer, pH 5.5, at a concentration of
12.5 mg/ml (1 mM). The arrow a indicates the
extremely high field-shifted methyl proton resonances. The arrow b indicates the down field-shifted
-proton resonances. The
protein concentration of the purified mBC domain was calculated from
the absorption at 280 nm (a value of 2.9). This value was calculated
using 1,576 M
cm
for tyrosine and 5,225 M
cm
for tryptophan
at 280 nm (Goodwin and Morton, 1946).
Figure 6: Far UV CD spectra of the wild-type and mutant BC domains. Far UV CD spectra of the wild-type, mutant C242A/C285A, and mutant W294A BC domains measured on a Jasco J-720 spectropolarimeter (Japan Spectroscopic) at various temperatures with solutions containing 0.12 mg/ml (10 µM) of the protein in 10 mM sodium phosphate buffer, pH 6.0, are shown. The optical path length was 1 mm for the far ultraviolet CD spectrum (190-250 nm). The concentration of the purified mBC domain was calculated as described in Fig. 3.
Figure 4:
Ligand binding activity of the mBC domain. Panel A shows an autoradiograph of the competitive ligand
binding of I-G-CSF and unlabeled G-CSF to the mBN and mBC
domains using chemical cross-linking, as described (Hiraoka et
al., 1994). Each reaction was performed with
I-G-CSF
(100,000 cpm, Amersham International), unlabeled G-CSF (Kirin Brewery),
and either the mBN or the mBC domain under the conditions indicated
above. Complexes of the mBN or the BC with
I-G-CSF were
treated with 1 mM dithiobis(succinimidyl propionate) (Pierce)
for cross-linking and were resolved by 0.1% SDS, 15% PAGE. The gel was
exposed to an imaging plate (Fuji imaging plate, Type BA 20X40; Fuji
Photo Film). The autoradiograph (upper part) of the gel from
the image analyzer data is shown (Bio-image analyzer BA100; Fuji Photo
Film), and the intensities of the bands corresponding to the
I-G-CSF bound to either the mBN (closed circles)
or the mBC (closed squares) domains were estimated (lower
part) as described (Hiraoka et al., 1994). The upper
arrowhead indicates the position of the G-CSF dimer. The middle arrowhead indicates the positions of the 1:1 complexes
of the mBN and the BC domains with G-CSF. The lower arrowhead indicates the position of G-CSF. The weak bands above 50 kDa are
due to contamination of the
I-G-CSF. The numbers show the sizes (kDa) of the marker proteins. The band intensities
of the mutant W294A
I-G-CSF complex (open
squares) in the presence of various amounts of unlabeled G-CSF are
plotted in the lower part. Panel B shows the
competition assay of the BC domain (0.5 µM) with 1.2
nM
I-G-CSF in the presence of 1 µM G-CSF, the BC domain of the human GH receptor (indicated as
hGHR-BC) (Asakura et al., 1994), RNaseA (Sigma), ovalbumin, or
bovine serum albumin (indicated as BSA, Pharmacia). The
mBC
I-G-CSF complex was treated with the chemical
cross-linker and was resolved by 0.1% SDS, 15% PAGE. The retained
radioactivity of the complex was counted as described (Hiraoka et
al., 1994).
Figure 5: Disulfide mapping of the mBC domain. Panel A shows the amino acid sequence of the mBC domain from residues 203-308, in addition to the GSS residues at the amino terminus. The boxes indicate the peptides generated by thermolysin digestion (TL11) and pepsin digestion after treatment with cyanogen bromide (BP17, BP22). Panel B shows the identification of the disulfide bonds in the mBC domain. Sequences in capital letters indicate the results of the Edman degradation of the non-reduced peptide. C denotes the di-PTH of cystine. (C) denotes a Cys residue that should be identified as the di-PTH of cystine, which was predicted from the known sequence but not observed. X denotes a Cys residue predicted from the known sequence but not observed. Numbers indicate the amino acid number of the mG-CSF receptor (Fukunaga et al., 1990a).
The BC domain specifically and stoichiometrically bound to
G-CSF. The tertiary folding of the BC domain was confirmed by NMR
spectroscopy. These data indicated that this domain (approximately 100
residues) folds discretely. NMR and far UV CD spectra indicated that
the BC domain has -structure. Analysis of the disulfide bonds of
the mBC domain of the G-CSF receptor showed that the 4 cysteine
residues of this domain formed two disulfide bonds. The structure of
the CRH region of the cytokine receptor was predicted to be
-strands held together by disulfide bonds (Bazan, 1990). These two
disulfide bonds (Cys
-Cys
,
Cys
-Cys
) were mapped between the B and E
-strands and between the C and F
-strands, respectively, on
the predicted topology (Fig. 7). These BC domain disulfide bonds
are not conserved in the cytokine receptor family (Bazan, 1990).
However, two disulfide bonds are essential for proper folding of the BC
domain of the G-CSF receptor. Miyazaki et al.(1991) determined
that the two Trp residues in the WSXWS motif are critical for
proper ligand binding to the interleukin-2 receptor
-chain, with
the use of a mutant cDNA transfection system in lymphoma cells.
Rozakis-Adcock and Kelly(1992) reported that substitution of the
WSXWS motif precluded high affinity ligand binding by the
prolactin receptor. However, our data suggest that the WSXWS
motif may play an important role in the proper folding of the BC domain
rather than in the formation of the ligand binding site. It is likely
that the disruption of the WSXWS motif leads to the formation
of an abnormally folded receptor and to a reduction in the ligand
binding activity. Recent x-ray crystallographic analyses of the GH and
prolactin receptors showed that the WSXWS motif (which is not
completely conserved in the GH receptor) is an irregular, extended
chain immediately preceding the following short
-strand and is
quite removed from the interfaces that contact ligand (de Vos et
al., 1992; Somers et al., 1994). Interestingly, it was
reported that the WSXWS box of the prolactin receptor is part
of a much larger, highly organized pattern, and the side chains of the
two Ser residues form hydrogen bonds to the main-chain atoms of a
neighboring
-strand (Somers et al., 1994). In the present
work, the removal of the side chains of the Ser residues in the two
mutants S295A and S298A caused lability of the mBC domain. Thus, it can
be speculated that these Ser side chains may form hydrogen bonds that
anchor the strands preceding the
-structure and stabilize the BC
domain. Yoshimura et al.(1992) suggested that the
WSXWS motif of the erythropoietin receptor is critical not
only for ligand binding but also for the ability of the receptor to
exit from the endoplasmic reticulum. Abundant work on secretory
proteins suggests that there is selective retention and/or degradation
of unfolded, misfolded, or aggregated proteins in the endoplasmic
reticulum (Pelham, 1989). These results are also consistent with our
results. The reduction of the yield of our precipitable mutants (S295A
and W297A) of the BC domain from the periplasmic fraction is compatible
with these results.
Figure 7:
Schematic drawing of the disulfide bonds.
The disulfide bonds of the mBC domain of the G-CSF receptor were mapped
on the -strand (A-G) topology maps predicted by
Bazan(1990). Numbers indicate the amino acid number of the
mG-CSF receptor (Fukunaga et al., 1990a). Thick circles indicate the amino acids corresponding to the WSXWS
motif.
The apparent K of
4-10
10
M indicates that the
BC domain plays a critical role in the recognition and the binding of
the ligand. This K
value is similar to that of
3-8
10
M exhibited by the
purified mBN domain of the G-CSF receptor (Hiraoka et al.,
1994). Both of the K
values of the BN and the BC
domains are relatively high as compared to the K
of 3-4
10
M exhibited
by the deletion mutant of the G-CSF receptor expressed on the surface
of murine myeloid cells, which contains both the BN and the BC domains
(Fukunaga et al., 1991). Our previous study indicated that the
BN domain also folds discretely (Hiraoka et al., 1994). These
results indicate that the CRH region of the G-CSF receptor is composed
of two small BN and BC domains, as predicted (Bazan 1990), and both
domains are required for high affinity ligand binding, such as
10
M. Probably, G-CSF recognition
occurs via a generic binding trough within the CRH region formed by the
BN and BC domains. Mutational and x-ray crystallographic analyses of
the human GH receptor indicated that two amino acid residues
(Trp
and Pro
) in its BN domain are critical
for human GH binding (Bass et al., 1991; de Vos et
al., 1992). Such important amino acid residues for ligand binding
were not detected in the BC domain of the human GH receptor, although a
substantial contact surface with the ligand exists in the BC domain.
However, most of the residues important for ligand binding in the GH
receptor and the interleukin-6 receptor could be located in or near the
hinge region of the two terminal domains (Bass et al., 1991;
Yawata et al., 1993).
In earlier work, Fukunaga et
al.(1991) reported the absence of detectable affinity of the
deletion mutants (Y(5-195)) of the mG-CSF receptor, which
contains the BC domain but lacks the immunoglobulin-like and the BN
domains, at the cell surface. Probably, the K
value of the mutant Y
(5-195) was below the level of
detection among the series of mutants analyzed with their system.
Recent analyses suggest that receptor oligomerization is the major
consequence of ligand binding, and a 2:1 receptor-ligand complex was
detected with the GH receptor (Cunningham et al., 1991; Fuh et al., 1992; de Vos et al., 1992). However, we did
not detect a 2:1 mBC domain
G-CSF complex by SDS-PAGE using
chemical cross-linking. This is reasonable, because our recent study
indicated that both the BC and the immunoglobulin-like domains, in
addition to the BN domain, are required for the oligomerization of the
G-CSF receptor (Hiraoka et al., 1995). Structural analysis of
the BC domain would aid further investigations of the mechanism and
function of this receptor. The size of the purified BC domain (12.5
kDa) is sufficiently small for NMR analysis.