(Received for publication, November 8, 1995; and in revised form, January 22, 1996)
From the
Residues in human leukemia inhibitory factor (hLIF) crucial for binding to both the human LIF receptor (R) and gp130 were identified by analysis of alanine scanning mutants of hLIF in assays for both receptor binding and bioactivity. The region of hLIF most important for binding to the hLIF-R is composed of residues from the amino terminus of the D-helix, carboxyl terminus of the B-helix, and C-D loop. This site forms a distinct surface at the end of the four-helix bundle in the tertiary structure of the closely related murine LIF. The two residues of hLIF that contribute the majority of free energy for hLIF-R binding, Phe-156 and Lys-159 are surrounded by other residues which have only a moderate impact. This arrangement of a few key residues surrounded by less important ones is analogous to the functional binding epitope of human growth hormone for its receptor. A second region of hLIF that includes residues from the carboxyl terminus of the D-helix and A-B loop also had a weak influence on hLIF-R binding. Residues in hLIF from both the A- and C-helices are involved in binding the gp130 co-receptor. Abolition of the gp130 binding site in hLIF created antagonists of LIF action.
Leukemia inhibitory factor (1, 2, 3) is a secreted cytokine that elicits
pleiotropic effects on a diverse range of cell types, these include
embryonic stem cells, primordial germ cells, neurons, adipocytes,
hepatocytes, and osteoblasts(4, 5) . In mice, gene
knockout experiments have demonstrated that LIF ()is
essential for embryonic implantation(6, 7) . In
contrast to the mild phenotype in mice lacking the LIF gene, the
targeted deficiency of the specific LIF receptor (LIF-R), results in
mice that have multiple placental, skeletal, neural, and metabolic
disorders, which cause perinatal death(8) . The biological
differences between these two genetic deficiencies is an outcome of the
use of the LIF-R for signal transduction by several ligands. The
cytokines known to bind to the LIF-R are included in a group that share
some biological properties with LIF: oncostatin M, ciliary neurotrophic
factor (CNTF), cardiotrophin (CT-1), interleukin-6 (IL-6), and
interleukin-11 (9, 10, 11, 12, 13, 14) .
The crystal structure of recombinant murine LIF (mLIF) has been
solved (15) . LIF has a four -helical bundle topology with
up-up-down-down helix orientation that has long crossover loops between
the first two and last two helices. The structure of LIF shows greatest
homology to granulocyte-colony-stimulating factor (G-CSF; (16) ), human growth hormone (hGH; (17) ) and the
recently determined CNTF(18) . These proteins all belong to the
hematopoietin cytokine family, which is characterized by the four-helix
bundle structure, but limited sequence homology between family
members(19, 20, 21) . The hematopoietin
cytokine family is divided into the short and long chain
subfamilies(21) . The long chain group of which both LIF and
hGH are members is characterized by helices of approximately 25
residues, the presence of short helical regions in the long loops, and
the complete absence of
strands. The canonical member of the
hematopoietin cytokine family and most characterized is hGH.
The co-crystal structure of hGH and its receptor(17) , together with extensive mutagenesis studies of both the ligand (22, 23) and receptor (24) , have defined both the signaling complex and binding surfaces of these molecules. The activated GHR is formed by homodimerization of two identical receptor subunits and a single hGH molecule(17, 25) . hGH uses two distinct sites to bind sequentially to the two growth hormone receptors, first via site I and secondarily to site II. The higher affinity GHR binding site on hGH, site I, involves residues in the carboxyl terminus of both the A- and D-helices and the A-B loop, whereas site II includes residues in both the A- and C-helices. In contrast to the ligand, almost identical residues on the GHR are used to bind both sites I and II on hGH.
Receptor homodimerization
leading to signal transduction as used by the GHR, erythropoietin
R(26) , and G-CSF-R (27) represents the simplest form
of receptor assembly. A more complex form of receptor association
involves heterodimerization, although the exact stoichiometry of
receptor components has not been determined for the majority of theses
complexes. Such complexes use a specific cytokine binding receptor for
each ligand and often share a common signaling chain among
several cytokines. This arrangement is demonstrated by interleukin 3,
granulocyte macrophage-colony stimulating factor (GM-CSF), and
interleukin-5 (IL-5), all of which use a common
receptor together
with a ligand-specific chain(28) . Similarly, the interleukins:
4, 7, 9, 15, and possibly 13 use the interleukin-2 (IL-2)
chain
together with a specific interleukin receptor binding component for
signal transduction(29) . Heterotrimeric receptor formation of
three different subunits is utilized for the association of the high
affinity IL-2 receptor(30) . The most complicated receptor
assembly so far characterized is that of the signaling unit of the
interleukin 6 receptor (IL-6-R). This complex involves receptor
heterodimerization, but forms a hexameric complex, consisting of two
molecules each of IL-6, IL-6-R, and gp130(31, 32) .
LIF transduces its biological signal via transmembrane receptors, of
which both low and high affinity forms have been characterized. The low
affinity species of the receptor (K10
M) is the single chain 190-kDa
LIF receptor(33) . Primary sequence comparison indicates that
the LIF-R is part of the hematopoietin receptor
family(20, 34) . The extracellular region of the LIF-R
is predicted to be composed of two hematopoietin domains separated by
an immunoglobulin module and three fibronectin type III repeats
proximal to the membrane(33) . Hematopoietin cytokine binding
domains have several primary sequence elements conserved in nearly all
members of the family, including several cysteine residues and the
motif Trp-Ser-X-Trp-Ser(20) . The structure of this
domain has been solved for both the prolactin receptor (35) and
GHR (17) , these structures are composed of two
-barrels
each consisting of seven anti-parallel
-strands.
The high
affinity signaling complex (K10
M) for LIF is formed by the
association of the LIF-R and a related member of the hematopoietin
receptor family, gp130(36, 37) . However, like many
other receptor complexes. the stoichiometry of components in the
activated complex is unknown. The gp130 receptor differs to the LIF-R
in that this receptor is predicted to have only a single hematopoietin
domain(33, 38) . Both LIF-R and gp130 are involved in
the high affinity receptor complex for cytokines related in structure
to LIF, these include oncostatin M(37, 39) ,
CT-1(14, 40) , and
CNTF(41, 42, 43) . The high affinity CNTF
receptor complex also requires the presence of a third component, the
CNTF receptor(44) , whereas, oncostatin M also uses an
alternative receptor to the LIF-R(45) . The gp130 receptor and
not the LIF-R is also involved as a co-receptor and signal transducer
for the cytokines, IL-6 (46, 47) and
IL-11(48, 49) . The common usage of gp130 and in some
cases also LIF-R for signal transduction by LIF, oncostatin M, CNTF,
CT-1, IL-6, and IL-11 explains their overlapping functional
characteristics. Both LIF-R and gp130 mediate intracellular signal
transduction via activation of cytoplasmic tyrosine kinases belonging
to the JAK and Src families(50, 51, 52) .
A clear understanding of how a particular cytokine interacts with its receptors is a crucial requirement for both comprehension of receptor activation and also the design of specific agonists and antagonists of cytokine action. Previous mutagenesis studies of LIF have utilized the observation that both murine and human LIF (hLIF) bind to the mLIF-R, but only hLIF will bind to the hLIF-R with high affinity. These studies suggested that residues in the C-D loop were important for this species specificity(15, 53) .
This study extended the previous work by evaluating the role of individual residues of hLIF to the binding to both hLIF-R and gp130. Human LIF mutants were analyzed both by direct binding assays to recombinant LIF-R-Fc and gp130-Fc and also for activity in a bioassay responsive to hLIF. Apart from revealing residues of hLIF critical for binding hLIF-R and gp130, abrogation of the gp130 binding site created antagonists of wild type hLIF.
The mature form of oncostatin M (57) was a kind gift from Dr. D Staunton; this protein was also expressed as a glutathione S-transferase fusion protein and purified as above.
The PCR primer matching the amino-terminal coding sequence also introduced an optimized Kozak sequence(59) , while the other primer created a splice donor sequence between codon 528 and the BamHI site. An identical approach was used to clone the human gp130 into the pIG plasmid, except the region amplified coded for residues 1-328. The PCR primers used were: amino terminus, TAGAAGCTTCCACCATGTTGACGTTGCAGACTTGGG; carboxyl terminus, ACGGATCCACTTACCTGTTGCTTTAGATGGTCTATCTT.
The nucleotide sequences of both constructs were confirmed by DNA sequencing with Sequenase (Amersham).
Binding studies to gp130-Fc were performed in manner similar to that
for LIF-R-Fc. These assays differed in that for gp130-Fc competition
binding, biotinylated oncostatin M was used instead of I-hLIF and the bound oncostatin M was detected by
incubation with a streptavidin-horseradish peroxidase conjugate
(Amersham). Specifically, after washing plates with PBS- 0.05% Tween
20, the wells were rinsed with PBS and then incubated with 100 µl
of streptavidin-horseradish peroxidase (1/1000 dilution) in PBS-1% BSA.
After again washing with PBS, the horseradish peroxidase was detected
by incubation with the chromagen OPD (orthophenylenediamine; Dako)
according to the manufacturer's instructions. Absorbance was read
at 492 nm in a 96-well plate reader (Anthos). All gp130-Fc binding
studies were performed in duplicate for at least two independent
experiments.
Figure 1:
Competitive inhibition of I-hLIF (0.165 nM) binding to LIF-R-Fc by hLIF or
hLIF mutants. Results are expressed as a ratio of counts bound at a
particular concentration of competitor [B] divided
by counts bound in the absence of competitor
[B
]. Values represent the mean of
triplicate samples, the S.E. for all points was less than 10% of the
mean. A:
, hLIF;
, hLIF F156A;
, hLIF
K159A. B:
, hLIF;
, hLIF-O1;
, hLIF-O3;
, hLIF-O5. C:
, hLIF;
, hLIF-O2;
,
hLIF-O4;
, hLIF-O6.
Figure 2:
Biological activity of hLIF or hLIF
mutants in the Ba/F3-hLIF-R/hgp130 assay. Results are expressed as the A value of cells assayed for proliferation by
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide(79) . Values represent the mean of triplicate samples,
the S.E. for all points was less than 10% of the mean. A:
, hLIF;
, hLIF F156A;
, hLIF K159A;
,
oncostatin M. B:
, hLIF;
, hLIF-O1;
,
hLIF-O2;
, hLIF-O3; *, hLIF-O4;
, hLIF-O5;
,
hLIF-O6.
Two mutants exhibited dramatic
reductions in the LIF-R-Fc binding assay, hLIF F156A and hLIF K159A ( Table 1and Fig. 1), the change in LIF-R-Fc affinity for
these two mutants was also paralleled by a large decrease in activity
in the proliferation assay ( Table 1and Fig. 2). A similar
reduction in LIF-R-Fc binding was also observed for mutants hLIF F156A
and hLIF K159A, when assayed for competition binding with I-hLIF on 293T cells transfected with the entire LIF-R
reading frame (data not shown). Despite the reduction in LIF-R-Fc
affinity in these mutants, no alteration in gp130-Fc affinity was
observed ( Table 1and Fig. 3), thus arguing that these
mutations were specific for LIF-R binding. Several other mutants
demonstrated significantly reduced binding to the LIF-R-Fc. These
included hLIF P51A, hLIF K153A, hLIF P106A, hLIF T150A, hLIF K158A, and
hLIF V175A (Table 1). Finally, several mutants showed only weak
(2-fold) reduction in LIF-R-Fc binding. These mutants included hLIF
D57A, hLIF K58A, hLIF D66A, hLIF K102A, hLIF D154A, hLIF K170A, and
hLIF A174Q (Table 1).
Figure 3:
Competitive inhibition of biotinylated
oncostatin M (0.8 nM) binding to gp130-Fc by hLIF or hLIF
mutants. Results are expressed as a ratio of biotinylated oncostatin M
bound att a particular concentration of competitor
[B] divided by biotinylated oncostatin M bound in
the absence of competitor [B]. Values
represent the mean of duplicate samples. A:
, hLIF;
, hLIF F156A;
, hLIF K159A;
, oncostatin M. B:
, LIF;
, hLIF-O1;
, hLIF-O3;
,
hLIF-O5. C:
, hLIF;
, hLIF-O2;
, hLIF-O4;
, hLIF-O6.
In general, the reduced LIF-R-Fc binding by the various mutants was also accompanied by a decrease in activity in the proliferation assay. However, the reduction in the LIF-R-Fc binding was usually greater than the reduction in the proliferation assay, even though the proliferation assay was able to detect lower LIF-R affinities. A lowered affinity for the LIF-R-Fc did not necessarily translate into a parallel reduction in biological activity, such as in mutant K153A. An analogous observation has been reported for mutants of IL-5(64) .
Of the mutants with significantly reduced LIF-R-Fc binding, all but three mutants had unchanged gp130-Fc affinity. The three mutants with altered gp130-Fc affinity all had less than 4-fold decreases in affinity. These mutants were hLIF P106A, hLIF K153A, and hLIF D154A (Table 1). The reduced affinity for gp130-Fc in the hLIF P106A mutant may be due to a change in the conformation of the B-C loop and thus may affect the packing of the C-helix, a contributor to gp130 binding (see below). For the hLIF K153A mutant, an additional mutation randomly introduced by PCR is present in the D-helix (G162D, Table 1), this extra change may alter the packing of the A-B loop against the D-helix and thus disrupt the structure. The presence of this second mutation confounds the contribution of Lys-153 to LIF-R binding.
Overall, the residues in hLIF that contribute the majority of free energy for binding to the LIF-R are located at the beginning of the D-helix (Phe-156 and Lys-159); these residues are spatially close together and have their side chains prominently exposed to the solvent in the hLIF structure (Fig. 5). Other residues that influence LIF-R binding map to the beginning of the D-helix (Lys-158), B-helix (Lys-102), and C-D loop (Thr-150); these amino acids are proximal to Phe-156 and Lys-159. Residues in the A-B loop (Asp-57, Lys-58) and the carboxyl terminus of the D-helix (Lys-170, Ala-174, Val-175) may form a second site of interaction with the LIF-R.
Figure 5:
Diagrammatic representation (Molscript; (80) ) of the hLIF structure depicting residues
important for binding to its receptors. The hLIF structure is very
similar to the mLIF molecule apart from a shift in which the A-B loop
crosses the D-helix. A, residues of hLIF with a major impact
on hLIF-R binding are displayed in red CPK and labeled.
Residues with a moderate impact on binding are displayed in magenta CPK. These residues are: Pro-51, Thr-150, Lys-158, and Val-175. N = amino terminus and C = carboxyl
terminus. The putative site I in hLIF would be centered around residue
Val-175, site III is centered on residues Phe-156 and Lys-159. B, residues of hLIF that when mutated collectively on either
the A- or C-helix caused reduced binding to gp130. Residues on the
A-helix are displayed in blue CPK, those on the C-helix are
displayed in yellow CPK. The residues in these two helices
compose the equivalent of site II in hLIF. N = amino
terminus and C = carboxyl
terminus.
Figure 4:
Antagonism of hLIF stimulation of the
Ba/F3-hLIF-R/hgp130 cell line by hLIF mutants impaired in gp130
binding. Human LIF mutants were titrated in the presence of a constant
concentration of hLIF (0.015 nM). Results are expressed as the A value of cells assayed for proliferation by
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide(79) . Values represent the mean of triplicate samples,
the S.E. for all points was less than 10% of the mean. A:
, no antagonist;
, hLIF-O1; │, hLIF-O3;
,
hLIF-O4;
, hLIF-O5. B:
, no antagonist;
,
hLIF-O2;
, hLIF-O6;
, hLIF-O4.
Activation of hematopoietin receptors requires at least the dimerization of two transmembrane receptors with cytoplasmic domains capable of signal transduction. The role of the ligand is to facilitate the oligomerization process. In the simplest example, signal transduction results from receptor homodimerization like the classical GHR. A more complex interaction involves receptor activation through heterodimerization; the activated human LIF receptor belongs in this category.
This study has identified residues of hLIF crucial for binding to both the LIF-R and gp130 by analysis of hLIF mutants in solid phase binding studies to both the LIF-R-Fc and gp130-Fc and also a bioassay responsive to LIF. The detectable binding of two different receptors to hLIF provided a convenient control for protein folding in mutant molecules. Thus, mutants that had decreased binding in one receptor but not the other strongly supported the idea that individual mutations caused local rather than global changes in structure.
The
hLIF residues Lys-159 and Phe-156 provide the majority of free energy
for binding to the LIF-R. These two residues are located at the
beginning of the D-helix, with their side chains adjacent and
prominently exposed to the solvent in both the mLIF molecule and the
recently determined hLIF structure (Fig. 5). ()Surrounding Lys-159 and Phe-156 are several other residues
in close proximity that influence the binding of hLIF to the hLIF-R (Table 1); collectively, these amino acids form a distinct region
at the end of the four-helix bundle in the tertiary structure of hLIF (Fig. 5). The arrangement of the LIF-R binding site of hLIF into
a few pivotal residues surrounded by ones of lesser importance was also
observed in the interaction of the hGH site I with the
GHR(23) .
The C-D loop and surrounding residues of hLIF have
been identified previously by chimera studies as being responsible for
the difference in binding affinity of murine and hLIF to the
hLIF-R(15, 53, 65) . In the most refined form
of this analysis, six mLIF residues were mutated to the equivalent hLIF
residues (E57D, T107S, Q112H, V113S, A155V, and R158K; (65) )
to give the mutated mLIF molecule high affinity binding to the hLIF-R.
That these residues were not identified in this investigation as
significant contributors of hLIF binding to hLIF-R may suggest these
residues prevent mLIF binding to the hLIF-R with high affinity by
either structural or chemical interference. This hypothesis is
supported by the observation that the human and mLIF structures show no
major shifts in the peptide backbone in the vicinity important for
LIF-R binding.
Other cytokines related to hLIF in primary sequence: mLIF, oncostatin M, CNTF, and CT-1 that can involve the hLIF-R in the activated receptor complex all have the hLIF residues Phe-156 and Lys-159 conserved(15, 40) , whereas IL-6 and IL-11 which do not bind the LIF-R lack these residues. No other residues of hLIF that influence binding to the LIF-R are conserved in all of these hLIF-R binding ligands. Thus, the equivalents of hLIF Phe-156 and Lys-159 in these other ligands may represent a common LIF-R binding motif. However, like mLIF, all of these molecules (excluding CT-1, which has yet to be examined) bind hLIF-R with significantly lower affinity than hLIF(15, 39, 41, 43, 53, 66) . Therefore, a LIF-R binding epitope involving homologues of Phe-156 and Lys-159 must be modulated by other residues in each individual cytokine.
The majority of hLIF residues identified in this study that were important for hLIF-R binding cluster at the end of the four-helix bundle. However, five residues with a weak influence on hLIF-R binding map to the carboxyl-terminal end of the D-helix and the A-B loop (Asp-57, Lys-58, Lys-170, Ala-174, Val-175). These residues may represent a second site of contact between hLIF and the hLIF-R. This second site could represent interaction with either another LIF-R molecule or another part of the LIF-R. Both these possibilities would be compatible with the use of a similar site on other long chain hematopoietin cytokines (see below and Table 2). The latter possibility is also in accordance with models proposed for LIF binding based on both the predicted two-hematopoietin domain structure of the LIF-R and competition binding characteristics of murine and hLIF (15, 67) . In these models, one LIF molecule is able to bind simultaneously to two distinct sites on a single LIF-R. However, the existence of a second site on LIF for LIF-R binding site requires further verification.
Residues of hLIF important for binding to gp130 were identified in a manner analogous to those involved in the LIF-R binding site, except that multiple simultaneous substitutions were used to locate the gp130 binding site. The interaction of LIF with gp130 is significantly weaker than the interaction with LIF-R which limits the ability to detect the influence of individual mutations with weak effects on the interaction with gp130. This analysis indicated that residues at the amino terminus of the A-helix contributed the majority of free energy for binding to gp130. In particular, all or a subset of the A-helix residues Gln-25, Ser-28, and Gln-32, participate directly in gp130 binding. The mutation of the C-helix residues: Asp-120, Ile-121, Gly-124, and Ser-127 also reduced gp130 binding, suggesting that all or some of these residues also interact with gp130. However, the role of individual hLIF amino acids in binding to gp130 cannot be determined for either the A- or C-helices without further mutagenesis investigations.
Antagonists
for several different four-helical bundle cytokines have been created
previously by mutations in ligand receptor binding surfaces. These
engineered antagonists include those for ligands which homodimerize
their receptors, for example hGH (68) and also cytokines which
involve heterodimerization in receptor activation such as interleukin
4(69) , IL-5(64) , and
GM-CSF(70, 71) . Similarly, in hLIF, abolition of the
gp130 binding site generates specific antagonists of hLIF in the
hLIF-responsive bioassay. The most active of these antagonists,
hLIF-O4, required 50-100-fold molar excess to inhibit 50%
activity of hLIF (Fig. 4). The requirement for an excess of
hLIF-O4 for significant inhibition is most likely a result of the
LIF-Rgp130 complex having approximately a 100-fold greater
affinity than the LIF-R for hLIF.
Generally, the antagonistic activity of the mutants impaired in gp130 binding was negatively correlated with the residual stimulation activity in the bioassay ( Table 1and Fig. 2and Fig. 4). However, the four mutants with essentially no bioactivity, yet nearly identical LIF-R-Fc binding, also showed distinct differences in antagonism. For example, hLIF-O4 (Q25A, S28A, and Q32A) was more effective than hLIF-O3 (Q25L, S28E, Q32A, S36A; Fig. 4). These differences in the antagonist activity of the hLIF mutants may be due to affinity differences for gp130, undetectable in the binding assay. Alternatively, a more complex interaction between the mutants and gp130 may explain these antagonism differences.
The pleiotropic nature of LIF and the number of ligands
that utilize the LIF-R will probably mean that LIF-R-specific
antagonists (the ability of these mutants to antagonize other LIF-R
binding ligands will be published elsewhere) ()will not be
of use therapeutically. However, the presence of soluble forms of gp130
in human serum that are able in vitro to inhibit
gp130-dependent cell stimulation may indicate that general antagonists
operate in vivo(72) . Moreover, the antagonists will
be of use in dissecting the complex and overlapping actions of
cytokines that use LIF-R and gp130 as receptors.
Finally, the results presented here strongly reinforce a pattern of receptor site usage among the long chain hematopoietin cytokines(73) . In this pattern, topologically conserved epitopes on different cytokines are used to bind cytokine receptors. To the initial paradigm of site I and II on hGH for GHR binding, a third receptor binding location has been definitively added by the mutational analysis of the known LIF structure presented here (site III; Fig. 5). Site III has also been recently predicted to bind a second molecule of gp130 on the modelled structure of IL-6(32) . The known receptor binding sites for other long chain cytokines are listed in Table 2. The common usage of receptor binding sites in this cytokine family suggests that other members such as CT-1, oncostatin M, and IL-11 will also use topologically similar epitopes.
The distribution of receptor binding
epitopes in non-overlapping regions of four helical bundle cytokines
provides a spatial explanation for multiple receptor engagement.
However, whether all complexes that involve heterotrimeric or even
heterodimeric receptor engagement use a single ligand in the activated
receptor or also involve higher order associations of ligands and
receptors such as in the IL-6 complex (31, 32) remains
to be resolved. This issue represents an important future goal for
understanding the LIFLIF-R
gp130 signaling complex
In summary, residues in hLIF that are important for binding both the LIF-R and gp130 were identified in this study, the disruption of the gp130 binding site resulted in the creation of LIF-R antagonists.