(Received for publication, March 16, 1995; and in revised form, July 9, 1995)
From the
The extracellular portion of the granulocyte colony-stimulating
factor (G-CSF) receptor has a mosaic structure of six domains (each
approximately 100 amino acid residues) consisting of an
immunoglobulin-like (Ig) domain, a cytokine receptor homologous region
subdivided into amino-terminal (BN) and carboxyl-terminal (BC) domains,
and three fibronectin type III repeats. In the present study, we
expressed the Ig-BN and the BN-BC regions and purified them to
homogeneity as monomers using G-CSF affinity column chromatography.
Using gel filtration high performance liquid chromatography, we
investigated the molecular composition of receptor-ligand complexes
formed between G-CSF and purified BN-BC or Ig-BN domains. In contrast
to the well characterized example of the human growth hormone (GH)
receptor, in which the BN-BCGH complex shows a 2:1
receptor-ligand complex stoichiometry, the BN-BC domain of the G-CSF
receptor formed a 1:1 complex. The isolated Ig-BN domain also formed a
1:1 complex with G-CSF. However, in the presence of both Ig-BN and
BN-BC domains, we detected a 1:1:1 Ig-BN
G-CSF
BN-BC complex
corresponding to the 2:1 receptor:ligand stoichiometry. These results
suggest that 1) the Ig domain and both the BN and the BC domains are
required for oligomerization of the G-CSF receptor, 2) G-CSF contains
two binding sites for its receptor, and 3) there are two ligand binding
sites on the G-CSF receptor, one site on the BN-BC domain and one on
the Ig-BN domain.
Ligand-induced receptor oligomerization has been proposed as the key mechanism of signal transduction for some families of single transmembrane receptors, such as cytokine receptors and tyrosine kinase-type receptors (Ullrich and Schlessinger, 1990; Wells et al., 1993; Heldin, 1995). In these models, the oligomerization of the extracellular regions, induced by ligand binding, is followed by the activation of their cytoplasmic regions. The extracellular regions of these receptors generally have a composite structure containing multiple domains (Ullrich and Schlessinger, 1990; Bazan, 1990; Miyajima et al., 1992; Heldin, 1995), which are presumed to play important roles in ligand-induced oligomerization. Investigation of the molecular properties of purified extracellular domains is a prerequisite for understanding mechanisms of receptor oligomerization that culminate in the transductions of external signals.
The
extracellular region (600 amino acid residues) of the granulocyte
colony-stimulating factor (G-CSF) (
)receptor is composed of
an immunoglobulin-like (Ig) domain, a cytokine receptor homologous
(CRH) region, and three fibronectin type III-like domains (Fukunaga et al., 1990b, 1990c). The Ig domain (
100 amino acid
residues) was originally defined by its homology to the immunoglobulin
superfamily (Williams and Barclay, 1988). Though a number of receptors,
including those for several cytokines, such as G-CSF, interleukin 6,
and ciliary neurotrophic factor, contain Ig domains (Fukunaga et
al., 1990b, 1990c; Larsen et al., 1990; Yamasaki et
al., 1988: Davis et al., 1991), little is known about
their function. A deletion derivate of the G-CSF receptor lacking the
Ig domain retained ligand binding activity, although the dissociation
constant (K
) value of the mutant was
10-20-fold higher than that of the intact receptor (Fukunaga et al., 1991). The CRH region (
200 amino acid residues)
was originally defined by its striking homology to the predicted ligand
binding domains of the receptors for various cytokines, such as
interleukins 2-7, granulocyte-macrophage colony-stimulating
factor, erythropoietin, growth hormone (GH), and prolactin (Bazan,
1990) receptors. The CRH region consists of an amino-terminal (BN;
100 amino acid residues) domain containing four conserved cysteine
residues and a carboxyl-terminal (BC;
100 amino acid residues)
domain containing a ``WSXWS'' motif (Bazan, 1990).
Earlier work has identified high affinity oligomers of the G-CSF
receptor on the surface of mouse myeloid leukemia cells (Fukunaga et al., 1990a), and mutational analyses showed that deletion
of the BN domain completely abolishes ligand binding activity (Fukunaga et al., 1991). As a first step toward studying its molecular
properties, we expressed the gene encoding the BN domain as a minimal
binding unit, using an Escherichia coli maltose binding
protein fusion system (Hiraoka et al., 1994a). This purified,
small domain still retained ligand binding activity but did not form
oligomers, such as dimers or tetramers (Hiraoka et al.,
1994a), suggesting that a larger binding unit is required for G-CSF
receptor oligomerization. As a second approach, we expressed a
three-domain binding unit consisting of Ig-BN-BC regions (
300
amino acid residues; indicated as Ig-CRH in Hiraoka et al.
(1994b)) using an insect Trichoplusia ni cell-baculovirus Autographa californica nuclear polyhedrosis virus system. The
purified Ig-BN-BC protein retained high ligand binding activity and
formed dimers and tetramers in the presence of G-CSF (Hiraoka et
al., 1994b). These studies indicated the involvement of the Ig or
BC regions in receptor oligomerization and suggested that expression of
tandem Ig-BN or BN-BC domains might permit functional dissection of the
oligomerization process.
In the present study, we describe
expression of the Ig-BN and the BN-BC domains of the G-CSF receptor in
insect cells using a baculovirus system. Isolated Ig-BN and BN-BC
domains did not form homo-oligomers in the presence of ligand, although
these products did form 1:1 binary complexes with G-CSF. However, the
combined Ig-BN and the BN-BC domains formed a 1:1:1
Ig-BNG-CSF
BN-BC complex, which is consistent with binding
of a single molecule of G-CSF ligand by a dimeric receptor.
Figure 1: Analysis of the purified Ig-BN and BN-BC domains of the G-CSF receptor. A, schematic representation of the Ig-BN, BN-BC, and Ig-BN-BC portions of the murine G-CSF receptor (Fukunaga et al., 1990b). The Ig, BN, and BC domains correspond approximately to exon 4, exons 5 and 6, and exons 7 and 8, respectively (Seto et al., 1992). The thin bars in the G-CSF receptor represent the conserved cysteine residues, and the thick bar represents the conserved WSXWS motif. Numbers indicate the amino acid number of the G-CSF receptor. B and C, 0.1% SDS-12.5% PAGE and Western blotting of the Ig-BN protein eluate from the TSKgel DEAE-2SW HPLC and the BN-BC protein eluate from the TSKgel G3000 SW gel filtration HPLC, respectively. The SDS-PAGE gels were stained with Coomassie Brilliant Blue (CBB). Western blotting was performed using anti-M1 serum (Fukunaga et al., 1991) as described (Hiraoka et al., 1994b). The numbers show the sizes (in kDa) of the marker proteins in lane M.
A DNA fragment corresponding to the BN-BC portion of the
murine G-CSF receptor (Fig. 1A) and its signal sequence
was generated from plasmid pBOSdIg, encoding a deletion mutant
G(5-84), which lacks Glu
-Ser
of the Ig domain (Fukunaga et al., 1991). To mutate
Cys
to Ser
in the residual portion of the Ig
domain of the G
(5-84) mutant, two polymerase chain reaction
fragments (fragment N and fragment C) were initially generated, using
pBOSdIg as the template. Fragment N corresponds to the G-CSF receptor
from the amino terminus to Val
, including the signal
sequence and the deleted part of the Ig domain. Primer 1 above was used
for the 5`-primer. The 3`-antisense primer has the sequence
5`-GGACGCCGATGTGTCCAGAGCTCTCCAGACTTCTGGGGAG-3`, which corresponds to
the sequence from Leu
to Ile
, and the
anti-codon Val
, in which the anticodon ACA for Cys
was replaced with AGA (underlined) for Ser. Fragment C
corresponds to the sequence from Leu
to Ala
of the deletion mutant cDNA G
(5-84), followed by a
stop codon. The 5`-primer has the sequence
5`-CTGGAGAGCTCTGGACACATCGGCGTCCAACTCCTG-3`, which contains the coding
sequence from Leu
to Ile
and from
Val
to Ala
, in which the codon TGT for
Cys
was replaced with TCT (underlined) for Ser. The
3`-antisense primer (primer 2) has the sequence
5`-GCTCTAGATTAGGCCTTCATGGTAGGCCTCA-3`, which corresponds to the
sequence for the carboxyl-terminal part of the BN-BC domain, terminated
at Ala
and followed by a stop codon and an XbaI
site (underlined). To amplify the full-length BN-BC DNA, small amounts
of fragments N and C were used as the template for a second round of
the polymerase chain reaction, using primer 1 as the 5`-primer and
primer 2 as the 3`-primer, respectively. Thus, the resulting fragment
contains the signal sequence
(Met
-Ser
), part of the Ig
domain (Ser-Gly
-His
-Ile
and
Val
-Gly
), and the BN-BC domain
(Tyr
-Ala
) of the murine G-CSF
receptor. After digestion with BamHI and XbaI, the
resultant fragment was used for the transfer vector, pAcBN-BC.
The transfer vectors pAcIg-BN and pAcBN-BC were obtained by the insertion of these amplified DNA fragments within the BamHI/XbaI sites of plasmid pVL1393 (Invitrogen), which carries part of the genome of the A. californica nuclear polyhedrosis virus. The BamHI/XbaI sites in pVL1393 are downstream of the polyhedrin promoter. The recombinant viruses AcIg-BN and AcBN-BC, carrying the Ig-BN and the BN-BC genes, were produced by in vivo homologous DNA recombination using the transfer vectors pAcIg-BN and pAcBN-BC, as described (Summers and Smith, 1988; Ota et al., 1991).
The molecular mass
of the Ig-BN protein, identified by SDS-PAGE and gel-filtration HPLC,
is larger than the molecular mass of 23 kDa deduced from the Ig-BN
protein sequence (Cys-Lys
) (Fukunaga et al., 1990b; Seto et al., 1992). The discrepancy
between the observed and predicted molecular masses is most likely due
to glycosylation. The Ig-BN protein has three potential Asn-linked
glycosylation sites, which are conserved between the murine and human
G-CSF receptors (Fukunaga et al., 1990b, 1990c). The 33-kDa
band identified by Western blotting of the culture fluid was not
detected when tunicamycin, a glycosylation inhibitor, was added to the
culture medium after infection, supporting the proposed glycosylation
of the Ig-BN protein. When Western blotting of the culture fluid was
performed under nonreducing conditions, we noted that the 33-kDa band
was less abundant and that most anti-M1 reactive material migrated at
more than 100 kDa. Presumably, most native Ig-BN molecules are secreted
into the culture fluid as large molecular weight aggregates due to the
effects of random disulfide bond formation, and only the 33-kDa
monomeric form, which bound to the G-CSF affinity column, was purified.
The far UV CD spectrum of the Ig-BN protein (Fig. 2A)
exhibited positive ellipticity at 230 nm and negative ellipticity
around 210 nm. The extracellular region of the human GH receptor (Fig. 2D) (Bass et al., 1991) showed a very
similar CD spectrum, suggesting that the purified Ig-BN protein has a
GH receptor-like structure. These results are consistent with the
spectra of the Ig-BN-BC region (Fig. 2C; Hiraoka et
al., 1994b) and the BN domain (Hiraoka et al., 1994a).
Figure 2:
Far-UV CD spectra of the Ig-BN, the BN-BC,
and the Ig-BN-BC regions of the G-CSF receptor and the BN-BC portion of
the GH receptor. The CD spectrum was measured on a Jasco J-720
spectropolarimeter (Japan Spectroscopic Co., Ltd.) at 20 °C with
solutions in 10 mM sodium phosphate buffer (pH 6.0) containing
0.093 mg/ml Ig-BN protein (A), 0.11 mg/ml BN-BC protein (B), and 0.12 mg/ml of Ig-BN-BC protein (C) (Hiraoka et al., 1994b) of the G-CSF receptor and 0.10 mg/ml of the
BN-BC protein of the GH receptor (D; indicated as hGH-R; Ota et al., 1991). The optical path length was
1 mm for the far ultraviolet CD spectra. The mean residue ellipticity,
[ø], has units of deg cm
dmol
. The purified protein concentrations were
calculated from the absorption at 280 nm (A
values of 1.3, Ig-BN; 2.2, BN-BC; and 1.9, Ig-BN-BC domains of
the G-CSF receptor, and 2.3, BN-BC portion of the GH receptor). 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).
A Scatchard analysis of the I-G-CSF binding data revealed that the BN-BC protein has
a K
of about 2.5
10
M. The far UV CD spectrum of the BN-BC protein (Fig. 2B) was similar to those of the Ig-BN domain and
the human GH receptor (Fig. 2D) (Bass et al.,
1991), suggesting that the BN-BC protein also has a GH receptor-like
structure. The amino-terminal sequence of the purified BN-BC protein
was determined. The predominant amino-terminal residue was Ala,
followed by the sequence Gly-Tyr-Pro-Pro. This amino-terminal residue
corresponds to Ala
of the
Ser
-Ile
,
Val
-Gly
interval (see underlines:
Ser
-Gly
-His
-Ile
and
Val
-Gln-Leu-Leu-Asp
-Gln-Ala-Glu-Leu-His-Ala
-Gly
)
derived from the Ig region. A minor (less than 10%, as estimated by gel
filtration HPLC), contaminating amino-terminal residue was Asp,
followed by the sequence Gln-Ala-Glu-Leu. This amino-terminal residue
corresponds to Asp
of the 16 residual amino acids from the
Ig domain. Thus, the expressed BN-BC protein contains the amino
terminus expected of the BN-BC domain; however, most of the short
Ig-derived sequence was removed during or after the processing of the
signal peptide. As a control, we expressed the BN-BC domain from
Tyr
to Ala
using an E. coli maltose
binding protein fusion system, as described (Hiraoka et al., 1994a). The E. coli purified BN-BC domain exhibited
almost the same K
for G-CSF and the same CD
spectrum as those of the purified BN-BC domain expressed using the
baculovirus system. (
)These results suggested that the
residual 16 amino acids of the Ig domain affect neither the K
nor the conformation of the BN-BC domain.
Figure 3:
Analysis of BN-BCG-CSF complexes by
gel filtration HPLC. The gel filtration profiles of various ratios of
the BN-BC protein to G-CSF, corresponding to 1:5 (line 1), 1:1 (line 2), 1:0.5 (line 3), 1:0.1 (line 4),
1:0 (line 5) (top to bottom tracing) are
shown. The protein concentrations of BN-BC (fixed at 1.6
µM) and G-CSF were calculated from the absorption at 280
nm (A
values of 2.2 and 0.81,
respectively) as described in Fig. 2. Protein mixtures were
equilibrated for 90 min at 20 °C in 20 mM sodium phosphate
buffer (pH 7.0) containing 0.2 M NaCl. Samples (200 µl)
were applied to a TSKgel G3000SW gel filtration HPLC (7.6 mm, inner
diameter,
60 cm; TOSO Co., Ltd) and were eluted with the same
buffer at 0.5 ml/min. Peaks were monitored for absorbance at 280 nm.
The elution positions of the BN-BC portion of human GH receptor, the
human GH, and their complexes estimated from Table 1are
indicated by arrows.
Fuh et al.(1990) expressed the BN-BC
portion of the GH receptor as a soluble protein using an E. coli secretion system. The molecular composition of the BN-BC portion
of the GH receptorGH complex established by gel filtration HPLC
showed a stoichiometry of 2:1 (Cunningham et al., 1991). As a
control to test the accuracy of our gel filtration assessment of
molecular mass, we also expressed the BN-BC portion of the GH receptor
using the insect-baculovirus system and attempted to induce the
formation of the 2:1 receptor
ligand complex. At a 1:0.5 ratio of
the BN-BC portion of the GH receptor to GH, all of the protein
chromatographed as a single peak, which corresponded to an 82-kDa
complex composed of two molecules of the BN-BC portion of the GH
receptor (30 kDa)/molecule of GH (22 kDa) (Fig. 4, line
3). When the ratio of the GH receptor to GH was decreased, a peak
appeared corresponding to a 1:1 complex of 52 kDa, and the 82-kDa peak
decreased in size (Fig. 4, lines 1 and 2).
These results confirmed that a 2:1 GH receptor to GH complex was
formed, and this complex dissociated to the 1:1 complex with an excess
of GH, as described (Cunningham et al., 1991; de Vos et
al., 1992; Fuh et al., 1992). These results also
indicated that the stoichiometry of the receptor BN-BC
ligand
complex differs between the G-CSF and the GH receptors, despite the
structural similarity between their BN-BC domains.
Figure 4:
Analysis of the BN-BC portion of the GH
receptor-GH complexes by gel filtration HPLC. The gel filtration
profiles of various ratios of the BN-BC portion of the GH receptor
(indicated as hGH-R) to GH, corresponding to 1:5 (line 1), 1:1 (line 2), 1:0.5 (line 3), 1:0.1 (line 4),
1:0 (line 5) (top to bottom tracing) are
shown. The protein concentrations of the BN-BC portion of the human GH
receptor (fixed at 1.3 µM) and GH (recombinant human GH;
Ota et al., 1991) were calculated from the absorption at 280
nm (A values of 2.3 and 0.80,
respectively) as described in Fig. 2. Protein mixtures were
equilibrated for 90 min at 20 °C in 20 mM sodium phosphate
buffer (pH 7.0) containing 0.2 M NaCl. Samples (200 µl)
were applied to the TSKgel G3000SW gel filtration HPLC and were eluted
with the same buffer at 0.5 ml/min. Peaks were monitored for absorbance
at 280 nm. The elution positions of bovine serum albumin (BSA;
67 kDa) and ovalbumin (ova; 43 kDa) are indicated by arrows.
The Ig-BN
(35-kDa) complex with G-CSF (19 kDa) was also established using gel
filtration HPLC (Fig. 5). At a 1:1 ratio of Ig-BN to G-CSF, all
of the protein chromatographed at 48 kDa (Fig. 5, line
2), corresponding to the size of a 1:1 complex (Table 1).
When the ratio of Ig-BN to G-CSF was 1:10, peaks corresponding to the
1:1 complex, as well as to free G-CSF, were present, and no peaks with
higher or lower molecular masses were detected (Fig. 5, line
3). These results show that the Ig-BN protein formed a stable 1:1
complex with G-CSF. When the Ig-BN protein was mixed with the BN-BC
protein in addition to G-CSF, a peak of 74 kDa appeared, with a
corresponding decrease in the 48-kDa peak (in ratios of
Ig-BN:G-CSF:BN-BC; 1:1:0, 1:1:0.5, 1:1:3; Fig. 5, lines
2, 4, and 5). This result is consistent with the
formation of a 1:1:1 Ig-BNG-CSF
BN-BC complex, as the
observed molecular mass of 74 kDa is quite close to the expected mass
of 81 kDa for a 1:1:1 ternary complex (Table 1). Components of
the ternary complex were confirmed by analysis of amino-terminal
residues released from the 74-kDa peak (data not shown). No
Ig-BN
BN-BC complex was formed in the absence of G-CSF (Fig. 5, line 6). The 35-kDa peak that would correspond
to release of the Ig-BN domain was not detected by gel filtration HPLC,
either upon addition of a small (1:1:0.5 ratio; Fig. 5, line
4) or an excess amount (1:1:3 ratio; Fig. 5, line
4) of the BN-BC domain. G-CSF was detected only in the 74-kDa peak
seen upon addition of an excess amount of the BN-BC domain (1:1:3
ratio; Fig. 5, line 5; Western blotting; data not
shown). These results suggested that the BN-BC protein did not compete
with the Ig-BN protein for binding to G-CSF. The formation of the
74-kDa peak and the retention of the Ig-BN domain in the complex were
confirmed by the addition of an excess amount of the BN-BC domain to
the purified Ig-BN
G-CSF complex, using gel filtration HPLC (data
not shown).
Figure 5:
Analysis of Ig-BNG-CSF and
Ig-BN
G-CSF
BN-BC complexes by gel filtration HPLC. The gel
filtration profiles of various ratios of the Ig-BN domain, G-CSF, and
the BN-BC domain, corresponding to 1:0:0 (lane 1), 1:1:0 (lane 2), 1:10:0 (lane 3), 1:1:0.5 (lane 4),
1:1:3 (lane 5), and 1:0:1 (lane 6) (top to bottom tracing) are shown. The protein concentrations of the
Ig-BN domain (fixed at 0.5 µM), the BN-BC domain, and
G-CSF were calculated from the absorption at 280 nm
(A
values of 1.3, 2.2, and 0.81,
respectively) as described in Fig. 2. Protein mixtures were
equilibrated for 90 min at 20 °C in 20 mM sodium phosphate
buffer (pH 7.0) containing 0.2 M NaCl. Samples (200 µl)
were applied to the TSKgel G3000SW gel filtration HPLC and were eluted
with the same buffer at 0.5 ml/min. Peaks were monitored for absorbance
at 280 nm. The elution positions of the BN-BC portion of human GH
receptor, the human GH, and their complexes estimated from Table 1are indicated by arrows.
The BN-BC domain of the human GH receptor forms a 2:1
receptorligand complex with the GH ligand, with consequent
dimerization of the GH receptor (Cunningham et al., 1991; de
Vos et al., 1992). We confirmed the existence of this 2:1
complex using GH receptor produced by a baculovirus system. Similar 2:1
receptor-ligand complexes have also been obtained using the receptors
for interferon-
and prolactin (Greenlund et al., 1993;
Hooper et al., 1993). Among the members of the cytokine
receptor family, these receptors, as well as the G-CSF receptor, seem
to function as homo-oligomers (Nagata and Fukunaga, 1991; Hiraoka et al., 1994b), supporting the view that the BN-BC domain of
the G-CSF receptor may form a 2:1 receptor-ligand complex.
Surprisingly, however, we found that the BN-BC domain of the G-CSF
receptor forms a 1:1 binary complex with its ligand G-CSF, suggesting
that the BN-BC domain of the G-CSF receptor alone is not sufficient for
G-CSF-induced oligomerization. Notably, extracellular portions of the
receptors for GH, interferon-
, and prolactin compose only the
BN-BC region (Bazan, 1990), while the G-CSF receptor contains Ig and
fibronectin type III domains in addition to the BN-BC functional unit.
In a previous study, we demonstrated that the purified Ig-BN-BC protein
existed as dimeric or tetrameric forms in the presence of G-CSF
(Hiraoka et al., 1994b). These results suggested the
possibility that the Ig domain might play an important role in
oligomerization of the G-CSF receptor.
Cunningham et al. indicated that the GH receptor contains two overlapping binding
sites that interact with two distinct sites on the GH protein, thereby
producing the 2:1 receptor-ligand complex (Cunningham et al.,
1991). In contrast, our results suggest that the BN-BC region of the
G-CSF receptor contains a single binding site for G-CSF, while the Ig
domain may provide a second ligand binding site. The purified Ig-BN
protein formed a stable 1:1 binary complex, indicating that the Ig-BN
region is also able to contribute to stable binding of G-CSF.
Furthermore, we detected the formation of a 1:1:1
Ig-BNG-CSF
BN-BC ternary complex upon addition of BN-BC
protein to the 1:1 Ig-BN
G-CSF complex. These results suggested
that two binding sites for G-CSF are found within the Ig-BN-BC region
and that, unlike the GH receptor paradigm, two different functional
units of the receptor molecule (both BN-BC and Ig-BN) are involved in
interactions with two distinct binding sites on the G-CSF ligand.
The apparent K of the BN-BC domain for G-CSF in
the present study is about 2.5
10
M. This value is similar to the K
of
about 3.7
10
M exhibited by the
deletion mutant of the G-CSF receptor expressed on the surface of a
murine myeloid cells that contains both the BN and the BC domains but
lacks the Ig domain (Fukunaga et al., 1991). We could not
measure the K
of the Ig-BN domain for G-CSF,
because of the low purification yield and weak affinity of the
anti-G-CSF receptor CRH antibody (anti-M1 serum; Fukunaga et
al., 1991) for the Ig-BN protein. Fukunaga et al. reported a K
value of about 1.1
10
M for a mutant G-CSF receptor expressing
the Ig and BN domains in addition to the fibronectin type III domains
but lacking the BC domain at the cell surface (Fukunaga et
al., 1991). Thus, it is likely that the affinity of the BN-BC
domain for G-CSF is higher than that of the Ig-BN domain.
Interestingly, in our study the Ig-BN domain was not released when the
BN-BC domain was incubated with the Ig-BN
G-CSF complex. This
finding is consistent with the idea that the binding site on the Ig-BN
domain is distinct from that of the BN-BC domain. We have reported
previously that the expressed Ig-BN-BC protein was purified primarily
as a dimer, using G-CSF affinity column chromatography, and that only
the dimeric Ig-BN-BC protein retained affinity for the ligand, termed
``high'' affinity (K
= about
10
M), while the monomeric Ig-BN-BC
protein showed reduced affinity, which was termed ``low'' (K
= about 2.5
10
M) (Hiraoka et al., 1994b). These data
supported the proposal that the Ig-BN-BC protein has two binding sites
and suggested that the two sites provided by each dimeric Ig-BN-BC
chain are required for high affinity binding of G-CSF. The apparent K
of the purified BN-BC domain (about 2.5
10
M) is nearly the same as that exhibited
by the monomeric Ig-BN-BC protein and corresponds to low affinity
ligand binding (Hiraoka et al., 1994b).
In our previous
work, we indicated that the Ig-BN-BCG-CSF complex was composed of
a dimeric Ig-BN-BC protein which forms at an extremely low
concentration of G-CSF, and that this dimer converted to a tetramer
with an increase in the concentration of ligand (Hiraoka et
al., 1994b). The stoichiometry of the tetrameric complex was
determined to be 4:4 Ig-BN-BC
G-CSF (Hiraoka et al.,
1994b). We could not estimate the stoichiometry of the dimeric complex
in that study because of the extremely low yield. In the present study,
we did not obtain any receptor-ligand complexes containing four
molecules of receptor (such as a 2:2:2 or 2:4:2
Ig-BN
G-CSF
BN-BC complexes, which would correspond to a 4:2
or 4:4 receptor-ligand stoichiometry), even at a high concentration of
G-CSF (data not shown). These data suggest that a minimal Ig-BN-BC
functional unit is required for the tetramerization. Fuh et
al. expressed chimeric GH and G-CSF receptor cDNAs in a leukemia
cell line and showed that the 2:1 GH receptor-GH complex, induced at a
low concentration of GH, was the active form for signal transduction,
whereas the 1:1 complex, induced at a high concentration of GH, was an
inactive form (Fuh et al., 1992). Thus, it is likely that the
receptor-ligand complex identified in the present study was
``frozen'' during purification as 2:1 G-CSF receptor-G-CSF
stoichiometry, which appeared as a 1:1:1 Ig-BN-BC
G-CSF
BN-BC
ternary complex (Fig. 6). The possibility remains that the G-CSF
receptor exists on the cell surface as a dimer and that higher
molecular weight aggregates, e.g. tetramers, constitute the
activated form of the receptor induced by G-CSF stimulation (Fukunaga et al., 1990a; Hiraoka et al., 1994b). However,
identification of a 1:1:1 Ig-BN
G-CSF
BN-BC ternary complex,
formed by two discrete ligand binding sites in separate functional
domains, suggests that the formation of a 2:1 G-CSF receptor-ligand
complex may play an important role in the function of the G-CSF
receptor, even if the dimerization of the G-CSF receptor is
insufficient to activate the receptor. Interestingly, a mutant G-CSF
receptor (G
(5-84)) containing the BN-BC and fibronectin type
III domains but lacking the Ig domain still retains weak signal
transduction activity (Fukunaga et al., 1991). Thus, if the
activated form of the G-CSF receptor is indeed a dimer, it is likely
that high concentrations of G-CSF can drive the G
(5-84)
mutant receptor into a dimeric form, at least to some extent, even in
the absence of the Ig domain. The mechanism and the meaning of the
conversion from dimeric to tetrameric forms of the G-CSF receptor are
still open questions. Further studies, including both mutational and
structural analyses, are required to understand the precise mechanism
of oligomerization and to correlate the observed oligomerization in
solution to the mitogenicity of G-CSF.
Figure 6:
Schematic representation of complexes of
the Ig-BN-BC, the BN-BC, and the Ig-BN domains of the G-CSF receptor
with G-CSF. A, each Ig-BN and BN-BC domain formed 1:1 binary
complexes with G-CSF. These Ig-BN and BN-BC regions form a 1:1:1
ternary complex with G-CSF in the presence of both the Ig-BN and the
BN-BC domains. B, thus, it is conceivable that the 2:1
Ig-BN-BCG-CSF complex may be formed at a low concentration of
G-CSF, and this 2:1 complex is converted to a 4:4 tetrameric complex
with an increasing concentration of G-CSF (see
``Discussion''; Hiraoka et al.,
1994b).