From the Departments of h Medicinal Chemistry and f Molecular Virology and Host Defence, GlaxoSmithKline, Collegeville, Pennsylvania 19426, a Structural Biology, i Protein Biochemistry, and j Gene Expression Sciences, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, and the Departments of e New Leads Discovery and c Transcription Research, Ligand Pharmaceuticals, San Diego, California 92121
Received for publication, September 9, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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Granulocyte colony-stimulating factor
regulates neutrophil production by binding to a specific receptor, the
granulocyte colony-stimulating factor receptor, expressed on cells of
the granulocytic lineage. Recombinant forms of granulocyte
colony-stimulating factor are used clinically to treat neutropenias. As
part of an effort to develop granulocyte colony-stimulating factor
mimics with the potential for oral bioavailability, we previously
identified a nonpeptidyl small molecule (SB-247464) that selectively
activates murine granulocyte colony-stimulating factor signal
transduction pathways and promotes neutrophil formation in
vivo. To elucidate the mechanism of action of SB-247464, a series
of cell-based and biochemical assays were performed. The activity of
SB-247464 is strictly dependent on the presence of zinc ions. Titration
microcalorimetry experiments using a soluble murine granulocyte
colony-stimulating factor receptor construct show that SB-247464 binds
to the extracellular domain of the receptor in a zinc
ion-dependent manner. Analytical ultracentrifugation
studies demonstrate that SB-247464 induces self-association of the
N-terminal three-domain fragment in a manner that is consistent with
dimerization. SB-247464 induces internalization of granulocyte
colony-stimulating factor receptor on intact cells, consistent with a
mechanism involving receptor oligomerization. These data show that
small nonpeptidyl compounds are capable of selectively binding and
inducing productive oligomerization of cytokine receptors.
Granulocyte colony-stimulating factor
(G-CSF)1 is the primary
cytokine controlling proliferation and differentiation of cells along
the granulocytic pathway (1). Lack of G-CSF results in a profound
neutropenia and increased susceptibility to infections (2). Recombinant
G-CSF is widely used to stimulate neutrophil production in patients
suffering from neutropenia as a result of chemotherapy or bone marrow
transplantation and is also used to mobilize hematopoietic precursor
cells into the periphery prior to leukopheresis (3, 4).
G-CSF is a member of the four-helix bundle family of cytokines and
binds to a type I cytokine receptor expressed on the surface of
neutrophils, granulocytic cells, and granulocytic precursors (5).
Binding of G-CSF to its receptor promotes oligomerization of receptor
chains, triggering the activation of JAK kinases associated with the
cytoplasmic region of the receptor (6-9). Activated JAKs then
phosphorylate specific tyrosine residues on a number of signaling
proteins including STAT3, STAT5, and SHC, which ultimately results in
changes in cell phenotype (7-9).
Type I cytokine receptors are defined by the presence of a CRH region,
which contains four conserved cysteine residues and a WSXWS
motif (8, 10, 11). The extracellular domain of some receptors, such as
those for erythropoietin and growth hormone, consist solely of a CRH
domain, which comprises the ligand binding site. Crystal structures
showing cytokines bound to CRH domains indicate that the CRH region
consists of two FN-III-like domains angled at ~90o with
respect to each other (12-14). Both structural and mutagenesis data
show that the cytokine interacts primarily with amino acids in loop
regions at the ends of the FN-III-like domains that project into the
space formed at the domain junction (14).
The extracellular domain of the G-CSF receptor also contains a CRH
region, which is separated from the membrane by three
FN-III-like repeats (15, 16). These repeats are not involved in
ligand binding but may play a role in stabilizing receptor dimers (16). A further Ig-like domain is attached to the N terminus of the CRH
region, completing the six-domain structure of the G-CSF receptor extracellular region. Biophysical characterization of soluble proteins
containing CRH domains alone or CRH- and Ig-like domains show that both
CRH and Ig-like domains play a role in binding G-CSF (17, 18). G-CSF
causes dimerization or higher order oligomerization of the receptor
subunits and of the intact G-CSF receptor extracellular domain
(17-20). The mechanism by which G-CSF promotes receptor dimerization
is not understood; however, a recent crystal structure of G-CSF bound
to the CRH domain of the receptor shows two G-CSF molecules interacting
with two CRH domains in a pseudosymmetric dimer (21). The major
contacts between each G-CSF molecule and the CRH domain occur at the
loop regions of a single CRH domain that project into the space between
the two FN-III-like repeats. As described above, this is similar to the way in which other cytokines of this class bind to their receptors and
is consistent with the predictions of mutagenesis experiments that had
previously identified amino acids in the CRH loop segments as important
for G-CSF binding (22). Each G-CSF monomer also participates in a minor
interaction with the second CRH monomer, and these interactions may
help stabilize the receptor dimer. Since the Ig-like domain is not
present in this structure, the details of its role in G-CSF binding and
receptor dimerization are not clear.
We have previously described the discovery and initial characterization
of a nonpeptidyl small molecule, SB-247464, that acts as a mimic of
G-CSF (23). This compound was discovered as part of a high throughput
screen designed to detect compounds that activate G-CSF signal
transduction pathways in a murine cell line. Like G-CSF, SB-247464
supports the formation of granulocytic colonies from primary murine
bone marrow in vitro. When injected into mice, SB-247464
causes increases in peripheral blood neutrophil counts and achieves an
efficacy equivalent to a saturating dose of recombinant G-CSF. Using
chimeric receptor constructs, we determined that expression of the
extracellular domain of the murine G-CSF receptor is required for
SB-247464 action, but the reason for this requirement was not
established. In this study, we have examined in more detail the mode of
action of the compound and its effect on the murine G-CSF receptor. Our
data show that SB-247464 interacts with the N-terminal half of the
extracellular domain of the receptor and promotes receptor
dimerization. Also, in contrast to G-CSF, we have found that the
binding and activity of SB-247464 is completely dependent on the
presence of zinc ions.
Luciferase Assay--
Cytokine-independent NFS60 cells
containing a stably integrated STAT-responsive luciferase reporter gene
that is induced by G-CSF have been described previously (23). The cells
were grown in RPMI containing 10% FBS and split to a density of 2 × 105/ml 2 days before the assay. Cells were transferred
to RPMI containing 0.5% FBS and 100-µl aliquots containing 1 × 105 cells incubated with G-CSF (10 ng/ml) or SB-247464 in
the presence of EDTA and metal chloride as indicated for 2.5 h.
SB-247464 was solvated in Me2SO. The final
Me2SO concentration in the assay was maintained at 0.3%.
At the end of the incubation period, the medium was removed from the
cells, which were then lysed, and luciferase levels were determined
using protocols and reagents obtained from Promega (Madison, WI) and a
Torcon luminometer. In some cases, results are graphed as relative
luciferase units, which are the numerical output of the luminometer.
Preparation of SB-247464 and0SB-250017--
For
SB-247464, synthesis was based on the condensation of 2,2'-pyridil with
2-guanidinobenzimidazole in the presence of base. Thus, a mixture of
2,2'-pyridil (15.8 g, 74.4 mmol) and 2-guanidinobenzimidazole (19.5 g,
111.7 mmol) in methanol (440 ml) was treated with a solution of sodium
hydroxide (2.97 g, 74.4 mmol) in 74 ml of water, and the resulting
mixture was left standing at room temperature for 4 days. The
crystalline material was filtered and dried under vacuum to yield
21.1 g of the title compound as off-white crystals (72%). mp
305-307 °C (dec); 010H NMR (300 MHz,
d060-Me020SO) 0
The compound SB-250017 was obtained from a pinacol-type rearrangement
as a minor component in the above reaction. Crystallization of the
mother liquors from the experiment above provided 500 mg of SB-250017
as the sodium salt. A second crop afforded a further 750 mg of compound
as the neutral form (overall yield of 6%). Structure was confirmed by
single crystal x-ray diffraction of the sodium salt material. mp
247-250 °C (dec); 1H NMR (300 MHz,
d6-Me2SO) Production of Soluble Murine G-CSF Receptor Proteins--
The
extracellular portion of the murine G-CSF receptor comprising amino
acids 1-626 (15) was expressed in CHOE1A cells as an Fc fusion protein
(G-CSFR-Fc). The N- and C-terminal three-domain fragments of the
receptor, IgCk1Ck2 (amino acids 1-332) and FnFnFn (amino acids
334-626), respectively, were also expressed in CHOE1A as Fc fusion
proteins but contained an FXa cleavage site to allow generation of
monomers by proteolytic removal of the Fc fragment (see Fig. 3). Fc
proteins were purified by protein G affinity chromatography followed by
size exclusion chromatography on a Superdex 200 column. Monomer
fragments were made from the dimeric Fc fusion proteins by incubation
with FXa, a second protein G column, and a sizing step with Superose
200. Purity of the products was evaluated by Coomassie Blue staining of
samples subjected to SDS-PAGE and was found to be better than 85% for
the G-CSFR-Fc protein and better than 90% for the monomeric IgCk1Ck2
and FnFnFn proteins. N-terminal sequences were confirmed for all the proteins.
Circular Dichroism Studies--
Circular dichroism measurements
were made with a Jasco J-710 CD spectropolarimeter at 0.3 mg/ml protein
concentration in a 0.1-cm water-jacketed cuvette. Wavelength scans were
made at 50 nm/min, and several spectra were recorded and averaged.
Isothermal Titration Calorimetry (ITC)--
Measurements were
made with a VP-ITC instrument (MicroCal, Inc., Northampton, MA) (24,
25). Dialyzed protein and ligand solutions were degassed for 10 min
prior to each titration. Receptor concentrations were determined by
absorbance at 280 nm using extinction coefficients calculated from the
amino acid sequences (26). Ligand concentrations were determined
gravimetrically. Titrations were carried out with injection volumes of
5-10 µl and a time interval between injections of 200 s. A
preliminary injection of 2 µl was made before each titration to
ensure the titrant concentration was at its loading value. Binding
isotherms were fitted by nonlinear regression using an assumed 1:1
stoichiometric model provided in the Origin ITC software (MicroCal)
(24). Although all of the reactions characterized involve multiple
equilibria (e.g. multiple SB-247464:zinc stoichiometric
species, overall binding reactions between SB-247464, zinc, and
receptor to form ternary complexes), there was not enough information
in the data to permit analysis by more complex models. Nevertheless,
the simple model used was found to describe all of the data to within
experimental uncertainty, and the resulting fitting parameters
(reported as empirical "observed" parameters) provide a useful
quantitative measure of overall binding affinity and thermodynamics for
comparative purposes. The empirical fitting parameters, reported as
observed parameters, are as follows: the binding molar ratio
(N), dissociation constant
(Kobs), and enthalpy change
( Analytical Ultracentrifugation--
Sedimentation equilibrium
data were measured on a Beckman XL-A analytical ultracentrifuge.
Samples were loaded in double sector cells with charcoal-filled epon
centerpieces and sapphire windows. Samples were centrifuged until
equilibrium was attained, as judged by an unchanging absorbance
versus radial position profile. The data were analyzed with
a single, homogeneous species model as in Ref. 27 with either a single,
homogeneous species model (Equation 1) or a monomer-dimer model
(Equation 2).
In the conditions of the present study, the solution density was
Detection of Activated STATs by Electrophoretic Mobility Shift
Assay--
NFS60 cells were grown and treated with G-CSF, SB-247464,
and/or SB-250017 using the conditions described above, except that 5 × 105 cells were used per treatment, and the
incubation period was 30 min. At the end of the incubation, cells were
lysed in 25 µl of lysis buffer (20 mM Hepes, pH 7.9, 300 mM NaCl, 10 mM KCl, 1 mM
MgCl2, 0.1% Triton X-100, 0.5 mM
dithiothreitol, 0.2 mM pefabloc, 20% glycerol, protease
inhibitors, 2 mM NaVO4) to give a whole cell
extract. Electrophoretic mobility shift assay reactions using 3 µl of
this extract and a labeled IRF-1 oligonucleotide STAT binding
site were performed as described (28).
Internalization Assays--
NFS60 cells were grown as described
above. 1 × 106 cells were incubated with G-CSF or
with SB-247464 for 2 h at 37 °C in RPMI containing 0.5% FBS.
Cells were then washed three times in ice-cold PBS and resuspended in
PBS. Cells were stained on ice by incubation with a 0.45 µg/ml
concentration of a G-CSF-phycoerythrin fusion protein (R & D Systems,
Minneapolis, MN) for 20 min and then washed three times in ice-cold PBS
and resuspended in PBS. Cell staining was quantitated using a
fluorescence-activated cell sorter (BD Biosciences). To test the
effect of SB-247464 on G-CSF-phycoerythrin binding, cells were briefly
incubated on ice with G-CSF or SB-247464 prior to washing and staining
with G-CSF-phycoerythrin as above.
Transient Transfection Assays--
The human and mouse G-CSF
receptor expression constructs have been described previously (23). The
chimeric receptors encode the following amino acids: M/H1, mouse amino
acids 1-612, human amino acids 612-707; M/H2, mouse amino acids
1-601, human amino acids 601-707; M/H3, mouse amino acids 1-594,
human amino acids 592-707. HepG2 cells were maintained in Dulbecco's
modified Eagle's medium containing 10% FBS. Cells were co-transfected
with the indicated G-CSF receptor expression construct and a luciferase reporter under the control of a multimerized STAT-binding element, 4×IRFtkluc (23). Cells were transfected in triplicate in 24-well plates using Superfect (Qiagen Inc., Chatsworth, CA), as instructed by
the manufacturer. 48 h after transfection, cells were treated with
10 ng/ml G-CSF or 1 µM SB-247464 for 4 h prior to
lysis and determination of luciferase activity as described above.
SB-247464 Activity Requires Zinc Ions--
Inspection of the
structure of SB-247464 showed a rigid 5,5 bicyclic system resulting
from the linkage of two opposing 2-guanidinobenzimidazole fragments.
These functional groups, together with the presence of the two
pyridines at the ring junctions, suggest a potential for the molecule
to interact with metal ions through coordination with the
sp2 lone pairs of three adjacent nitrogen atoms, as shown
in Fig. 1. They also allow for the
formation of a dimer of SB-247464 linked by two metal ions. Both this
type of tridentate coordination and dimer formation have
actually been demonstrated by x-ray crystallographic studies of a
number of metal complexes derived from
SB-247464.2 To test for an
effect of metal ion concentration on compound activity, we incubated
SB-247464 in tissue culture medium containing EDTA, a metal
chelator, at various concentrations, and assayed for compound activity
using a G-CSF-inducible reporter cell line. Incubation of this cell
line with G-CSF or with SB-247464 alone causes a readily detectable
increase in luciferase expression (Fig.
2A); for SB-247464, maximal
expression of luciferase took place at a concentration of 1 µM. Activation of luciferase by 1 µM
SB-247464 was progressively decreased in the presence of increasing
concentrations of EDTA (Fig. 2B). In this concentration range (0.3-100 µM), EDTA had no effect on luciferase
induction in response to G-CSF, indicating that the elimination of the
SB-247464 response is not due to a nonspecific effect on luciferase
expression or cell viability (Fig. 2C). The activity of 1 µM SB-247464 in the presence of 50 µM EDTA
could be recovered by titrating in additional zinc (II) ions (Fig.
2D). The reduction in luciferase activity at higher
concentrations of Zn(II) is due to nonspecific toxicity of Zn(II) at
these concentrations. Titrating in other metal ions such as Mn(II) and
Fe(II) (Fig. 2D) or Cu(II), Ni(II), or Co(II) (data not
shown) did not restore the original activity elicited by SB-247464,
indicating a selective requirement of zinc ions for the G-CSF mimetic
activity of SB-247464. Direct evidence for this zinc ion dependence is
presented below.
SB-247464 Binds to the Murine G-CSF Receptor--
Our previous
data demonstrated a requirement for expression of the murine G-CSF
receptor for SB-247464 activity (23). This could be because the
compound directly interacts with the receptor and triggers signal
transduction or because it binds to a nonreceptor target that requires
the presence of receptor to activate the G-CSF signaling cascade. In
order to distinguish between these possibilities and to investigate the
role of zinc ions, we conducted biophysical studies to test for direct
binding of SB-247464 to the receptor. The complete extracellular domain
of the murine G-CSF receptor was expressed in mammalian cells as a
secreted Fc domain fusion protein (G-CSFR-Fc). N- and C-terminal halves of the extracellular domain, designated IgCk1Ck2 and FnFnFn,
respectively, were also expressed as Fc fusion proteins. These two
fusions incorporated a protease cleavage site, allowing isolation of
monomeric IgCk1Ck2 and FcFcFc fragments (Fig.
3A). Molecular masses of the
purified proteins were measured by matrix-assisted laser
desorption/ionization mass spectrometry to be 238,258 Da (G-CSFR-Fc),
44,939 Da (IgCk1Ck2), and 47,087 Da (FnFnFn). Secondary structures of
the IgCk1Ck2-Fc and FnFnFn-Fc constructs were found to be indicative of
well folded proteins having predominantly
Fig. 4 shows titration calorimetry
measurements for SB-247464 in the presence of ZnCl2 alone
and in the presence of ZnCl2 plus the G-CSFR-Fc construct.
As shown in Fig. 4A, binding of SB-247464 to zinc(II) is
very tight (apparent Kd < 50 nM) and
readily detected from the observed binding enthalpy change of the
overall reaction. The molar ratio of ~1:1 is consistent with
preliminary x-ray crystallographic studies of the complexation of
SB-247464 with metal ions, which showed formation of a quaternary complex consisting of two molecules of SB-247464 wrapped around two
metal ions. To detect binding of SB-247464 to the receptor we compared
the titrations of SB-247464 into zinc both in the presence and absence
of G-CSFR-Fc construct. As seen in Fig. 4B, titration in the
presence of G-CSFR-Fc resulted in a more exothermic process, with
binding heats several kcal/mol higher than those obtained in its
absence. The apparent Kd of the process was also
higher (~1.2 µM), indicating lower affinity of the
SB-247464-zinc complex for the construct, compared with the
binding of SB-247464 and zinc. Because of the presence of multiple
equilibria, the apparent Kd derived from these data
does not represent the true Kd for binding of the
SB-247464-zinc complex to G-CSFR-Fc (see "Experimental
Procedures"). Control experiments, either with the isolated Fc domain
or with other Fc fusion proteins, demonstrated that the binding
thermochemistry observed with G-CSFR-Fc is due to interactions with the
GCSF-R portion of the construct. In the absence of zinc (Fig.
4C), binding of SB-247464 to G-CSFR-Fc could not be
detected, indicating that, as in the reporter-gene experiments above,
the interaction is mediated by zinc ions.
Evidence that SB-247464 Promotes Receptor Oligomerization--
The
presumed mechanism for activating G-CSF receptor on the cell surface is
induced self-association (9, 19-21). To further investigate the
binding and agonist mechanism of SB-247464, we conducted analytical
ultracentrifugation experiments on monomeric forms of the N- and
C-terminal three-domain fragments of the receptor shown in Fig. 3. Fig.
5A shows analytical
ultracentrifugation data for both constructs in the presence and
absence of SB-247464; ZnCl2 was used in these experiments.
In the IgCk1Ck2 case (left), the addition of SB-247464
changed the shape of the sedimentation curve and increased the weight
average mass from 46 to 66 kDa. In the FnFnFn case (right),
the curves without and with SB-247464 had the same shape, differing
only in amplitude, and the weight average masses were within error
unchanged (54 versus 51 kDa, respectively). These
mass-average molecular weights for FnFnFn are slightly higher than
those determined by mass spectrometry. The difference may arise from
uncertainties in the partial specific volume of the protein, the
solution density, and potential deviations from the single exponential
model used for curve fitting in Fig. 5A. Curve-fitting the
FnFnFn data in Fig. 5A to an oligomerization model did yield
an improvement in goodness of fit.
The residuals for the single-species fit (Equation 1) to the IgCk1Ck2
data were random in the absence of SB-247464 but showed a systematic
trend in its presence, indicating lack of goodness of fit and hence the
presence of multiple species. In contrast, SB-247464 had no discernible
effect on the pattern of residuals for the FnFnFn fits.
The IgCk1Ck2 centrifuge data with SB-247464 can be interpreted by
fitting with assumed monomer-oligomer equilibrium models. The best fit
curve in Fig. 5B represents a monomer-dimer model, with a
predicted Kd of 15 µM and improved
randomness of the residuals ( A Nonsymmetrical Analogue of SB-247464 Acts as an
Antagonist--
The C2-symmetrical structure of SB-247464
suggests that its capacity to dimerize the G-CSF receptor may result
from its ability to recognize and bind to two receptor chains
simultaneously, an interaction mediated by zinc ions. This model would
predict that a derivative of SB-247464 with only single metal-binding
and receptor recognition sites might be able to bind to, but not
dimerize, the receptor and could potentially act as an antagonist of
SB-247464. The imidazolinone SB-250017 (Fig. 1), with only one of the
guanidinobenzimidazole groups of SB-247464, was identified as such a
compound. Fig. 6A shows
titration calorimetry data for SB-250017 binding to zinc (left) and zinc in the presence of G-CSFR-Fc
(right). Here the binding enthalpy changes are smaller than
with SB-247464, but the data readily demonstrate that SB-250017 can
bind G-CSFR-Fc through an interaction also mediated by zinc(II).
Analytical ultracentrifugation data demonstrated that SB-250017 did not
induce self-association of the IgCk1Ck2 monomer (Fig.
6B).
Treatment of NFS60 cells with SB-247464 or G-CSF for 15 min causes
activation of STAT3 and STAT5 that can be detected by electrophoretic mobility shift assay using a STAT-binding oligonucleotide (7, 28). This
assay was used to test the effect of SB-250017 on the activation of
G-CSF signal transduction pathways by SB-247464. NFS60 cells were
incubated with 1 µM SB-247464 in the presence of either
no SB-250017 or increasing concentrations of SB-250017. The medium was
supplemented with 30 µM ZnCl2 to provide an
excess of zinc ions in the assay. SB-250017 alone did not cause
activation of STATs, consistent with its inability to dimerize G-CSF
receptor chains. However, SB-250017 significantly antagonized the
activation of STATs by SB-247464 (Fig.
7). Increasing the concentration of ZnCl2 in the assay to 100 µM did not relieve
this repression, indicating that antagonism was not due to preferential
chelation of zinc ions by SB-250017.
Internalization of G-CSF Receptors by SB-247464--
Dimerization
or oligomerization of receptors by cytokines and growth factors leads
to internalization of receptors over a period of several hours (29). To
seek additional evidence that SB-247464 affects the oligomerization
state of G-CSF receptors in living cells, a receptor internalization
assay was performed. NFS60 cells were preincubated with SB-247464 or
G-CSF at 37 °C, conditions that allow receptor internalization to
occur (30-32). Following this preincubation, levels of residual G-CSF
receptor at the cell surface were measured by staining with a
fluorescently labeled G-CSF fusion protein. Fig.
8A shows that preincubation with SB-247464 at concentrations between 0.03 and 1.0 µM
caused a reduction in cell surface G-CSF receptors compared with cells preincubated with medium alone. Interestingly, preincubation
with higher concentrations of SB-247464 caused a smaller loss of cell surface receptors. As expected, preincubation with G-CSF caused a
reduction in measured cell surface receptors, with a maximal effect
occurring at 1 ng/ml. The assay was then performed at 4 °C, a
temperature that is nonpermissive for receptor internalization (30-32). Under these conditions, SB-247464 did not affect the ability of the fluorescent G-CSF fusion protein to bind to NFS60 cells, indicating that the reduction in cell staining seen at 37 °C is not
due to SB-247464 interfering with the binding of G-CSF to the receptor
(Fig. 8B).
Additional Requirement for Transmembrane Sequences--
Our
previous work with chimeric human/murine G-CSF receptors had suggested
that the C-terminal region of the murine extracellular domain appeared
to be required for the activity of SB-247464 (23). This inference was
based on the observation that a chimeric receptor in which the
N-terminal half of the human G-CSF receptor extracellular domain was
replaced with the equivalent region of the murine receptor was not
responsive to SB-247464. We constructed additional mouse/human G-CSF
receptor chimeras to examine this requirement further. These constructs
were transfected into HepG2 cells and tested for their ability to
activate a STAT-responsive reporter after G-CSF or SB-247464 treatment.
The results are shown in Fig. 9. As
previously reported (23), the murine G-CSF receptor is activated by
SB-247464, whereas the human receptor is not. A chimeric receptor in
which the extracellular domain and first 11 amino acids of the
transmembrane domain are murine in origin is activated efficiently by
both G-CSF and SB-247464. However, a chimeric receptor in which the
junction between murine and human sequences is at the start of the
predicted transmembrane domain is only marginally activated by
SB-247464, although activation by G-CSF is unaffected. A third chimeric
receptor in which all of the transmembrane domain and the first 9 amino acids of the extracellular domain are human behaves similarly.
SB-247464 was originally discovered by virtue of its ability to
activate G-CSF signal transduction pathways and was subsequently found
to be a selective mimic of G-CSF both in vitro and in
vivo (23). These are novel activities for a low molecular weight, nonpeptidyl molecule, and it was therefore of considerable interest to
determine both the molecular target for SB-247464 and its mechanism of
action. The cell-based assay used to identify SB-247464 can in
principle detect compounds acting at many points in the G-CSF signaling
cascade (33). Our initial characterization of SB-247464 indicated a
requirement for murine G-CSF receptor expression for compound activity.
However, it was not established whether the receptor was in fact the
direct target for SB-247464 or whether it played an indirect role in
compound activity.
Here we show that SB-247464 interacts with soluble extracellular domain
fragments derived from the murine G-CSF receptor using both isothermal
calorimetry and analytical ultracentrifugation. This strongly suggests
that the extracellular domain of the receptor is the molecular target
for SB-247464. The primary binding site for SB-247464 maps to the
N-terminal half of the extracellular domain, comprising the Ig-like
domain and the two FN-III-like domains of the CRH region. This is the
same fragment of the receptor that binds to G-CSF (17, 18, 22).
However, SB-247464 appears to be unable to compete with G-CSF for
binding to the intact G-CSF receptor on NFS60 cells, suggesting that it
binds to this region of the receptor at a site not involved in
interactions with G-CSF. This is in contrast to the peptide mimics of
erythropoietin and thrombopoietin and a recently described
nonpeptidyl mimic of erythropoietin, which compete with the
natural cytokine for receptor binding (34-36).
The in vitro binding studies show that SB-247464
does not bind to receptor in the absence of zinc ions. This
explains the requirement for zinc ions for compound activity in the
cell-based luciferase assay. Serum contained in the tissue culture
media contains sufficient zinc ions to promote compound
activity, but in biochemical assays it must be supplied
exogenously. Isothermal calorimetry provides direct evidence that
SB-247464 binds zinc ions, presumably via the two metal chelation sites
present in the compound. The precise role of zinc ions in promoting
compound binding is not known. In addition to their role in catalysis
and stabilizing protein structure, zinc ions are known to mediate certain protein-protein interactions via coordination of amino acids in
both proteins (37-39). In this case, the zinc ion may be coordinated
to both SB-247464 and amino acids in the receptor, directly mediating
compound binding. Alternately, zinc ions may indirectly promote
compound binding by stabilizing the conformation or oligomerization
state of SB-247464 that constitutes the receptor binding species.
Analysis of mouse/human chimeric G-CSF receptor constructs shows that
amino acids in the murine transmembrane domain are required (but not
sufficient) for efficient activation of the receptor by SB-247464.
SB-247464 does not efficiently activate chimeric G-CSF receptors in
which the transmembrane domain is entirely human in origin, even if
they contain the binding site for the compound. The murine
transmembrane domain may transmit or permit a conformational change in
response to compound that is impeded by the human receptor
transmembrane domain. In contrast, the response of chimeric G-CSF
receptors to G-CSF is not influenced by the species derivation of the
transmembrane domain.
The analytical ultracentrifuge experiments demonstrate that SB-247464
causes oligomerization of the G-CSF receptor extracellular domain. This
observation provides an explanation for the ability of the compound to
mimic the activities G-CSF in cellular and in vivo assays.
Dimerization of the G-CSF receptor has been shown to be sufficient to
trigger signal transduction, an observation that is generally true for
homodimeric receptors of this family (40-45). Dimerization or higher
order oligomerization of G-CSF receptor extracellular domain fragments
in the presence of G-CSF has been reported in a number of biochemical
studies and is evident in the crystal structure of the receptor CRH
domain bound to G-CSF (17-21). The symmetry of SB-247464 allows the
molecule to simultaneously bind to two zinc ions in a
C2-symmetrical fashion. This property of SB-247464 suggests
a model where the resulting SB-247464(Zn)2 ternary complex
interacts with two different receptor chains, promoting their
dimerization on the surface of the cell. This model is consistent with
the observation that the related compound SB-250017, which lacks the
symmetry elements of SB-247464 and contains a single zinc-binding site,
can bind to the receptor but cannot dimerize it. Instead, SB-250017
acts as an antagonist of SB-247464, although it has no effect on the
activation of receptor by G-CSF. This latter observation also suggests
that the details of receptor dimerization by SB-247464 and G-CSF
differ, perhaps indicating that there are multiple pathways that lead
to receptor activation.
Internalization of G-CSF receptors by SB-247464 provides additional
evidence that this compound induces receptor clustering. The shape of
the dose-response curve in this assay warrants discussion. Increasing
concentrations of SB-247464 initially induce greater levels of receptor
down-regulation, up to 1 µM. Higher concentrations reduce
receptor internalization, however. This inverted bell-shaped dose-response curve mirrors the bell-shaped dose-response curve seen
with SB-247464 in the luciferase assay. Increasing concentrations of
SB-247464 initially cause increased levels of luciferase expression, but higher concentrations elicit lower levels of luciferase expression. This effect is not due to compound toxicity. If SB-247464 induces receptor dimerization by binding to two receptor chains, these dose-response curves can be explained by self-antagonism of the compound at high concentrations due to occupation of all available receptor chains by a single molecule of SB-247464. A similar phenomenon has been described for growth hormone and erythropoietin (42, 43) but
has not been observed for G-CSF
(46),3 again suggesting that
SB-247464 activates the receptor in a novel manner. A detailed review
of this effect and its implications has been presented by Whitty and
Borysenko (47).
Our data do not exclude an alternate model for SB-247464-mediated
receptor dimerization, in which the compound binds to a single receptor
chain and induces a conformational change that favors dimerization.
However, this model does not readily explain the dose-response curves
described above. Structural data showing the details of SB-247464
interaction with the receptor will allow a more complete understanding
of the mechanism of action of this compound and may provide insight
into differences between G-CSF- and SB-247464-mediated receptor activation.
These results show that low molecular weight compounds can selectively
bind, dimerize, and activate relatively large receptors that are
normally activated by protein ligands. Since compounds such as
SB-247464 are readily amenable to optimization through standard
medicinal chemistry techniques, it should be possible to develop small
molecule, orally bioavailable drugs with the potential to replace
injectable recombinant proteins such as cytokines.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0
11.5 (br s, NH, 2 H), 10.0 (br s, NH, 2 H), 8.6 (br s, NH, 2 H), 8.38 (d, J 0 = 4.2 Hz, 2 H), 7.55 (t, J 0 = 7.8 Hz, 2 H), 7.29 (d, J 0 = 7.8 Hz, 2 H), 7.27-7.21
(m, 4 H), 7.14 (br s, 2 H), 6.98 (dd, J 0 = 5.8, 3.2 Hz, 4 H); 0130C NMR (100.6 MHz,
d060-Me020SO) 0
0
159.6 (2 C), 157.8 (2 C), 155.6 (2 C), 148.9 (2 CH), 137.4 (4 C), 136.8 (2 CH), 123.9 (2 CH), 122.8 (2 CH), 120.7 (4 CH), 113.0 (4 CH), 85.7 (2 C); MS (electrospray ionization) m0/z 0 527 [M + H]0+0; Anal. Calcd for
C0280H0220N0120.
0 02/3H020O: C, 62.44; H, 4.37. N, 31.21; Found: C, 62.72; H, 4.08; N, 30.86.
11.8 (br s, NH, 1 H),
9.5 (br s, NH, 1 H), 8.6 (br s, NH, 2 H), 8.48 (d, J = 4.8 Hz, 2 H), 7.77 (t, J = 7.8 Hz, 2 H), 7.51 (d,
J = 7.8 Hz, 2 H), 7.38 (br s, 2 H), 7.29 (dd,
J = 7.8, 4.8 Hz, 2 H), 7.10 (br s, 2 H); MS
(electrospray ionization) m/z 370 [M + H]+.
Hobs). Due to the very low solubility of
SB-247464, experiments were conducted in buffer containing 3%
Me2SO plus 30% sucrose, which was found empirically to
enable SB-247464 to be dissolved up to 150 µM.
(Eq. 1)
Here
(Eq. 2)
= (M
|
)
2(r2
r
,r represents the
absorbance at the detection wavelength
and radial position
r, cm is the concentration of monomer at
the meniscus, and
is the molar extinction coefficient/mol of
monomer at wavelength
. M and |
are the molecular
weight and partial specific volume of a monomer subunit,
is the
solvent density,
is the angular velocity, rm is
the radial position at the meniscus (in centimeters), R is
the universal gas constant, T is the absolute temperature,
and offset is a constant offset fitting parameter.
= 1.1173 g/ml, and |
values were calculated from sequence as 0.7364 and 0.7387 ml/g, respectively, for IgCk1Ck2 and FnFnFn monomer constructs. The floating parameters in Equation 1 are M, cm, and offset.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Structure of metal complexes of SB-247464 and
SB-250017. SB-247464 is arbitrarily shown with the exocyclic imine
bonds in the trans configuration. Coordination of metal ions
by the chelating nitrogens in the compounds is indicated by the
dashed lines. Solid lines
indicate potential additional coordination of the metal ion to other
ligands (L). Note that a dimeric complex of SB-247464 held
together by two metal ions can also be formed.
View larger version (27K):
[in a new window]
Fig. 2.
The activity of SB-247464 in a
STAT-dependent reporter assay requires zinc ions.
A, induction of luciferase activity by SB-247464 in tissue
culture medium. NFS60 cells containing a STAT-responsive
reporter were incubated with SB-247464 in RPMI containing 0.5% FBS.
After 2.5 h, luciferase activity in the cells was determined.
Bars, S.D. (n = 3). This panel is
adapted from our previous work (23). RLU, relative
luciferase units. DMSO, Me2SO. B and
C, effect of EDTA on luciferase induction by SB-247464 and
G-CSF in tissue culture medium. Cells were incubated with 1 µM SB-247464 (B) or 10 ng/ml G-CSF
(C) in RPMI containing 0.5% FBS and the indicated
concentration of EDTA. After 2.5 h, luciferase activity in the
cells was determined. Bars, S.D. (n = 3).
D, effect of metal chlorides on SB-247464 activity in the
presence of EDTA. Cells were incubated with 1 µM
SB-247464, 50 µM EDTA in RPMI containing 0.5% FBS.
Various metal chlorides were added as indicated (ZnCl2
(Z), MnCl2 (M), and FeCl2
(F)). After 2.5 h, luciferase activity in the cells was
determined. Results are graphed as percentage of luciferase activity
relative to a control in the absence of any metal chloride.
Bars, S.D. (n = 3).
-sheet structure (minimum
ellipticity at 215 nm) by far UV circular dichroism spectroscopy (Fig.
3B). Both constructs were stable against thermal
denaturation up to at least 45 °C.
View larger version (27K):
[in a new window]
Fig. 3.
Domain structure and characterization of
murine G-CSF receptor extracellular domain fusion proteins.
A, domain structure of murine G-CSF receptor extracellular
domain fusion proteins produced for binding and dimerization studies.
Individual domains are labeled Ig (immunoglobulin-like),
Ck1, Ck2, FN-III (fibronectin type
III), and Fc (immunoglobulin constant region, used for
protein purification) to indicate their known or predicted structural
classification. The conserved cysteine residues and the
WSXWS motif characteristic of type I cytokine receptors are
indicated by c and a vertical black
bar, respectively. A factor Xa cleavage site engineered into
the IgCk1Ck2-Fc and FnFnFn-Fc constructs is indicated by the
horizontal black bar. Incubation of
these proteins with Factor Xa and isolation of the cleaved receptor
fragments yields the monomeric proteins shown on the right.
B, far UV circular dichroism spectra of IgCk1Ck2-Fc and
FnFnFn-Fc constructs at 20 °C shown as observed ellipticity
versus wavelength. Open circles,
IgCk1Ck2-Fc; solid circles, FnFnFn-Fc.
Inset, thermal denaturation curves shown as ellipticity at
215 nm versus temperature. Both constructs are thermally
stable to at least 45 °C. Conditions were as follows: 10 mM HEPES, pH 7.3, 150 mM NaCl, 30% sucrose,
0.1 cm path length, and 0.3 mg/ml receptor protein.
View larger version (19K):
[in a new window]
Fig. 4.
SB-247464 binds to the extracellular domain
of the murine G-CSF receptor in a zinc ion-dependent
manner. Titration microcalorimetry data for mixing SB-247464 with
11 µM ZnCl2 (A), 11 µM ZnCl2 plus 5 µM murine
G-CSFR-Fc (B), and 5 µM muG-CSFR-Fc plus 300 µM EDTA (C). Top panels,
unprocessed microcalories/s versus time data. At every
injection of 10 µl of 150 µM SB-247464 into the
calorimeter cell, a spike is observed. The integrated area of each
spike yields the amount of heat for each titration increment. The
lower panels show the amount of heat observed at
each injection normalized per mole of SB-247464 injected
versus the accumulated molar ratio of SB-247464 added per
zinc ion. Curves are the best fit to an assumed single binding site
model, with best fit apparent parameters equal to
Kobs = 0.047 ± 0.020 µM and
Hobs =
4.4 ± 0.1 kcal/mol
(left) and Kobs = 1.2 ± 0.1 µM and
Hobs =
11.9 ± 0.2 kcal/mol (right). Conditions were as follows: 10 mM HEPES, pH 7.3, 30 °C, 150 mM NaCl, 30%
sucrose, and 3% Me2SO.
View larger version (28K):
[in a new window]
Fig. 5.
SB-247464 changes the oligomerization state
of the murine G-CSF receptor IgCk1Ck2 protein solution.
A, analytical ultracentrifugation analysis of the effect of
SB-247464 on the weight average molecular masses of murine IgCk1Ck2
(left) and FnFnFn (right) monomers. In each case,
the data and best fit curves (Equation 1) are shown in the
lower panels, and the residuals for the fits are
shown in the upper panels. Open
circles, in the presence of 10 µM SB-247464;
solid squares, in its absence. Weight average
masses of the constructs were determined as follows: 45.7 ± 0.4 kDa (IgCk1Ck2 alone), 66.3 ± 1.7 kDa (IgCk1Ck2 + SB-247464),
53.7 ± 0.1 kDa (FnFnFn alone), and 51.3 ± 0.1 kDa (FnFnFn + SB-247464). Conditions were as follows: 3 µM receptor
protein, 15 µM ZnCl2, 10 mM
HEPES, pH 7.3, 20 °C, 150 mM NaCl, 30% sucrose, 1%
Me2SO, and 20,000 rpm. B, interpretation of
IgCk1Ck2 and SB-247464 centrifugation data with a monomer-dimer
equilibrium model. Best-fit curve (Equation 2) is shown in the
lower panel. The upper
panel shows residuals for fitting to the monomer-dimer model
(solid squares) together with residuals for a
single-species model (Equation 1, open circles).
Deconvolution of the fitted curve into predicted monomer (M)
and dimer (D) species is shown as the dashed
curves as labeled. The dotted line is
the best fit absorbance offset (Equation 2).
2 decreasing from 5.09 × 10
3 for single species fit to 3.74 × 10
3). The data could also be reasonably fitted by a
monomer-trimer model (
2 = 3.88 × 10
3), but models proposing the existence of tetramers
(
2 = 5.06 × 10
3) or higher oligomers
could be excluded as judged by lack of goodness of the fits. More
thorough experimental testing of the monomer-dimer model by
centrifugation was not carried out due to prohibitively long
equilibration periods required in 30% sucrose solvent (3 days per
condition). Overall, the data suggest that SB-247464 binds
preferentially to an oligomeric form of IgCk1Ck2 relative to monomer,
thus driving self-association of the receptor. This association was
shown to be a noncovalent interaction, since analysis by nonreducing
SDS-PAGE did not reveal higher molecular weight species expected from a
disulfide cross-linking mechanism (data not shown).
View larger version (28K):
[in a new window]
Fig. 6.
SB-250017 binds to the murine G-CSF receptor
extracellular domain in a zinc ion-dependent manner but
does not induce changes in oligomerization state. A,
titration microcalorimetry data for mixing SB-250017 with 11 µM ZnCl2 (left) and 11 µM ZnCl2 plus 4 µM G-CSFR-Fc
(right). Curves are the best fit to an assumed single
binding site model, with best fit apparent parameters equal to
Kobs = 0.098 ± 0.027 µM and
Hobs =
2.80 ± 0.05 kcal/mol
(left), and Kobs = 0.444 ± 0.076 µM and
Hobs =
4.40 ± 0.08 kcal/mol (right). Conditions were as in Fig. 4.
B, analytical ultracentrifugation analysis of the effect of
SB-250017 on the weight average molecular masses of murine IgCk1Ck2.
The data and best fit curve (Equation 1) are in the lower
panel, and the residuals are in the upper
panel. Open circles, in the presence
of 10 µM SB-250017; solid squares,
in its absence. Weight average masses in the absence and presence of
SB-250017 were determined as 45 ± 0 kDa and 42 ± 0 kDa,
respectively. Conditions were as follows: 3 µM receptor
protein, 15 µM ZnCl2, 10 mM
HEPES, pH 7.3, 20 °C, 150 mM NaCl, 30% sucrose, 3%
Me2SO, and 20,000 rpm.
View larger version (32K):
[in a new window]
Fig. 7.
SB-250017 antagonizes activation of STATs by
SB-247464. NFS60 cells were incubated with 1 µM
SB-247464 in the presence of the indicated concentrations of SB-250017
in RPMI containing 0.5% FBS and 30 µM ZnCl2.
After 30 min, cells were lysed, and activated STATs were detected using
a radiolabeled IRF-1 STAT binding oligonucleotide in an electrophoretic
mobility shift assay. The autoradiograph of the resulting gel is shown,
together with a graph of the amount of STAT-DNA complexes formed
derived from quantitation of the gel.
View larger version (19K):
[in a new window]
Fig. 8.
Incubation of NFS60 cells with SB-247464 at
37 °C causes a reduction in cell surface G-CSF receptor levels.
A, NFS60 cells were incubated with the indicated
concentration of SB-247464 (left panel) or G-CSF
(right panel) at 37 °C in RPMI containing
0.5% FBS. After 2 h, cells were cooled on ice, washed, and
stained with a fluorescent G-CSF fusion protein. Stained cells were
detected using a fluorescence-activated cell sorter. B,
NFS60 cells were incubated with SB-247464 or G-CSF on ice in RPMI
containing 0.5% FBS and then stained and analyzed as above.
View larger version (23K):
[in a new window]
Fig. 9.
Transmembrane domain sequences are required
for efficient activation of G-CSF receptor by SB-247464.
A, structure of chimeric mouse/human G-CSF receptors. Human
sequences are shown in white; mouse sequences are in
black. The number at the junction of each
chimeric receptor refers to the murine amino acid at the junction. The
amino acid sequence of the mouse and human G-CSF receptors with
junction points indicated by arrows is also shown. The
predicted location of the transmembrane domain is indicated by the
box. B, activity of the chimeric receptors and
control mouse and human receptors in response to G-CSF or SB-247464
treatment. HepG2 cells were cotransfected in triplicate with the
indicated receptor expression vectors and a STAT-responsive luciferase
reporter, 4×IRFtkluc. Induction of luciferase activity compared with
untreated controls (-fold induction) is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
FOOTNOTES |
---|
* 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.
b Present address: Bristol Myers Squibb PRI, Princeton, NJ 08543.
d Present address: Deltagene, 4570 Executive Dr., San Diego, CA 92121.
g Present address: Centocor, 200 Great Valley Pkwy., Malvern, PA 19355.
k Present address: Sugen Inc., 230 E. Grand Ave., South San Francisco, CA 94080.
l To whom correspondence should be addressed: Exelixis Inc., 170 Harbor Way, South San Francisco, CA 94080. Tel.: 650-837-7064; Fax: 650-837-8181; E-mail: plamb@exelixis.com.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M209220200
2 M. L. Doyle, A. E. Baker, M. R. Brigham-Burke, K. J. Duffy, R. M. Keenan, and J. I. Luengo, manuscript in preparation.
3 S.-S. Tian, unpublished result.
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ABBREVIATIONS |
---|
The abbreviations used are: G-CSF, granulocyte colony-stimulating factor; G-CSFR, granulocyte colony-stimulating factor receptor; CRH, cytokine receptor homology; FN-III-like, fibronectin type-III-like; FBS, fetal bovine serum; IRF, interferon response factor; ITC, isothermal calorimetry; PBS, phosphate-buffered saline; STAT, signal transducers and activators of transcription; MS, mass spectrometry.
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---|
1. | Demitri, G. D., and Griffin, J. D. (1991) Blood 78, 2791-2808[Medline] [Order article via Infotrieve] |
2. |
Lieschke, G. J.,
Grail, D.,
Hodgson, G.,
Metcalf, D.,
Stanley, E.,
Cheers, C.,
Fowler, K. J.,
Basu, S.,
Zhan, Y. F.,
and Dunn, A. R.
(1994)
Blood
84,
1737-1746 |
3. |
Welte, K.,
Gabrilove, J.,
Bronchud, M. H.,
Platzer, E.,
and Morstyn, G.
(1996)
Blood
88,
1907-1929 |
4. | Tabbara, I. A., Ghazal, C. C, and Ghazal, H. H. (1997) Cancer Invest. 15, 353-357[Medline] [Order article via Infotrieve] |
5. | Shinjo, K., Takeshita, A., Ohnishi, K., and Ohno, R. (1997) Leuk. Lymphoma 25, 37-46[Medline] [Order article via Infotrieve] |
6. | Nicholson, S. E., Oates, A. C., Harpur, A. G., Ziemiecki, A., Wilkes, A. F., and Layton, J. E. (1994) Proc. Natl. Acad. Sci. 91, 2985-2988[Abstract] |
7. |
Tian, S-S.,
Lamb, P.,
Seidel, H. M.,
Stein, R. B.,
and Rosen, J.
(1994)
Blood
84,
1760-1764 |
8. | Ihle, J. N., Witthuhn, B. A., Quelle, F. W., Yamamoto, K., and Silvennoinen, O. (1995) Annu. Rev. Immunol. 13, 369-398[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Avalos, B. R.
(1996)
Blood
88,
761-777 |
10. | Bazan, J. F. (1990) Proc. Natl. Acad. Sci. 87, 6934-6938[Abstract] |
11. | Moutoussamy, S., Kelly, P. A., and Finidori, J. (1998) Eur. J. Biochem. 255, 1-11[Abstract] |
12. | de Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992) Science 255, 306-312[Medline] [Order article via Infotrieve] |
13. | Syed, R. S., Reid, S. W., Li, C., Cheetham, J. C., Aoki, K. H., Liu, B., Zhan, H., Oslund, T. D., Chirino, A. J., Zhang, J., Finer-Moore, J., Elliot, S., Sitney, K., Katz, B. A., Matthews, D. J., Wendoloski, J. J., Egrie, J., and Stroud, R. M. (1998) Nature 395, 511-516[CrossRef][Medline] [Order article via Infotrieve] |
14. | Kossiakoff, A. A., and De Vos, A. M. (1998) Adv Protein Chem. 52, 67-108[Medline] [Order article via Infotrieve] |
15. | Fukunaga, R., Ishizaka-Ikeda, E., Seto, Y., and Nagata, S. (1990) Cell 61, 341-350[Medline] [Order article via Infotrieve] |
16. | Fukunaga, R., Ishizaka-Ikeda, E., Pan, C-X., Seto, Y., and Nagata, S. (1991) EMBO J. 10, 2855-2865[Abstract] |
17. |
Hiraoka, O.,
Anaguchi, H.,
Yamasaki, K.,
Fukunaga, R.,
Nagata, S.,
and Ota, Y.
(1994)
J. Biol. Chem.
269,
22412-22419 |
18. | Hiraoka, O., Anaguchi, H., Asakura, A., and Ota, Y. J. Biol. Chem. 270, 25928-25934 |
19. | Horan, T. P., Martin, F., Simonet, L., Arakawa, T., and Philo, J. S. (1997) J. Biochem. (Tokyo) 121, 370-375[Abstract] |
20. | Horan, T., Wen, J., Narhi, L., Parker, V., Garcia, A., Arakawa, T., and Philo, J. (1996) Biochemistry 35, 4886-4896[CrossRef][Medline] [Order article via Infotrieve] |
21. | Aritomi, M., Kunishima, N., Okamoto, T., Kuroki, R., Ota, Y., and Morikawa, K. (1999) Nature 401, 713-717[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Layton, J. E.,
Iaria, J.,
Smith, D. K.,
and Treutlein, H. R.
(1997)
J. Biol. Chem.
272,
29735-29741 |
23. |
Tian, S-S.,
Lamb, P.,
King, A. G.,
Miller, S. G.,
Kessler, L.,
Luengo, J. I.,
Averill, L.,
Johnson, R. K.,
Gleason, J. G.,
Pelus, L. M.,
Dillon, S. B.,
and Rosen, J.
(1998)
Science
281,
257-259 |
24. | Wiseman, T., Williston, S., Brandts, J. F., and Lin, L.-N. (1989) Anal. Biochem. 179, 131-137[Medline] [Order article via Infotrieve] |
25. | Doyle, M. L. (1999) in Current Protocols in Protein Science (Chanda, V., ed) pp. 20.4 1-24, John Wiley and Sons, Inc., New York |
26. |
Pace, C. N.,
Vajdos, F.,
Fee, L.,
Grimsley, G.,
and Gray, T.
(1995)
Protein Sci.
4,
2411-2423 |
27. | Doyle, M. L., and Hensley, P. (1997) Adv. Mol. Cell. Biol. 22, 279-337 |
28. |
Tian, S-S.,
Tapley, P.,
Sincich, C.,
Stein, R. B.,
Rosen, J.,
and Lamb, P.
(1996)
Blood
88,
4435-4444 |
29. | Sorkin, A., and Waters, C. M. (1993) Bioessays 15, 375-382[Medline] [Order article via Infotrieve] |
30. | Khwaja, A., Carver, J., Jones, H. M., Paterson, D., and Linch, D. C. (1993) Br. J. Haematol. 85, 254-259[Medline] [Order article via Infotrieve] |
31. |
Hunter, M. G.,
and Avalos, B. R.
(1999)
Blood
93,
440-446 |
32. |
Ward, A. C.,
van Aesch, Y. M.,
Schelen, A. M.,
and Touw, I. P.
(1999)
Blood
93,
447-458 |
33. | Seidel, H. M., Lamb, P., and Rosen, J. (2000) Oncogene 19, 2645-2656[CrossRef][Medline] [Order article via Infotrieve] |
34. | Wrighton, N. C., Farrell, F. X, Chang, R., Kashap, A. K., Barbone, F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K., and Dower, W. J. (1996) Science 273, 458-463[Abstract] |
35. |
Cwirla, S. E.,
Balasubramanian, P.,
Duffin, D. J.,
Wagstrom, C. R.,
Gates, C. M.,
Singer, S. C.,
Davis, A. M.,
Tansik, R. L.,
Mattheakis, L. C.,
Boytos, C. M.,
Schatz, P. J.,
Baccanari, D. P.,
Wrighton, N. C.,
Barret, R. W.,
and Dower, W. J.
(1997)
Science
276,
1696-1699 |
36. |
Qureshi, S. A.,
Kim, R. M.,
Konteatis, Z.,
Biazzo, D. E.,
Motamedi, H.,
Rodrigues, R.,
Boice, J. A.,
Calaycay, J. R.,
Bednarek, M. A.,
Griffin, P.,
Gao, Y. D.,
Chapman, K.,
and Mark, D. F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12156-12161 |
37. | Berg, J. M., and Shi, Y. (1996) Science 271, 1081-1085[Abstract] |
38. | Coleman, J. E. (1998) Curr. Opin. Chem. Biol. 2, 222-234[CrossRef][Medline] [Order article via Infotrieve] |
39. | Coleman, J. E. (1992) Annu. Rev. Biochem. 61, 897-946[CrossRef][Medline] [Order article via Infotrieve] |
40. | Heldin, C-H. (1995) Cell 80, 213-223[Medline] [Order article via Infotrieve] |
41. |
Elliot, S.,
Lorenzini, T.,
Yanagihara, D.,
Chang, D.,
and Elliot, G.
(1996)
J. Biol. Chem.
271,
24691-24697 |
42. |
Schneider, H.,
Chaovapong, W.,
Matthews, D. J.,
Karkaria, C.,
Cass, R. T.,
Zhan, H.,
Boyle, M.,
Lorenzini, T.,
Elliott, S. G.,
and Giebel, L. B.
(1997)
Blood
89,
473-482 |
43. | Ishizaka-Ikeda, E., Fukunaga, R., Wood, W. I., Goeddel, D. V., and Nagata, S. (1993) Proc. Natl. Acad. Sci. 90, 123-127[Abstract] |
44. | Cunningham, B. C., Ultsch, M., De Vos, A. M., Mulkerrin, M. G., Clauser, K. R., and Wells, J. A. (1991) Science 254, 821-825[Medline] [Order article via Infotrieve] |
45. |
Remy, I.,
Wilson, I. A.,
and Michnick, S. W.
(1999)
Science
283,
990-993 |
46. |
Young, D. C.,
Zhan, H.,
Cheng, Q-L.,
Hou, J.,
and Matthews, D. J.
(1997)
Protein Sci.
6,
1228-1236 |
47. | Whitty, A., and Borysenko, C. W. (1999) Chem. Biol. 6, R107-R118[Medline] [Order article via Infotrieve] |