From the
By rational mutagenesis, receptor-specific functional analysis,
and visualization of complex formation in solution, we identified
individual amino acid side chains involved specifically in the
interaction of ciliary neurotrophic factor (CNTF) with CNTFR
Ciliary neurotrophic factor (CNTF)
CNTFR
Soluble
CNTFR
In addition to the well characterized
interaction of CNTF with CNTFR
The prediction that CNTF, LIF, IL-6, and several
other ligands have a similar four helix bundle fold
(15) and the
realization that they share common receptors have led to an intense
search for residues involved in receptor recognition by sequence
alignment, computer modeling, and rational mutagenesis. However, the
limited sequence identity among these proteins, the absence of
three-dimensional structures, and the difficulties of systematic
mutagenesis have not allowed precise localization of receptor epitopes
beyond the identification of a few residues critical for biological
activity and/or receptor binding.
Here, we describe the
identification on the surface of CNTF of a cluster of residues that
constitutes an epitope for CNTFR
A different result was obtained with the D30Q
protein. Addition of sCNTFR
Overall, two distinct types of CNTF variants emerged
from this analysis. The first,
To critically test this conclusion, we examined the
location of these amino acids on the three-dimensional structure of
human CNTF
(13) . Residues 25, 28, 63, 64, 74, and 177 are indeed
exposed on the surface of the molecule and thus are capable of direct
contact with CNTFR
It is worth noting that among the more than 90
other amino acid substitution and deletion mutants
()
Three of the amino acids in the CNTFR
To summarize the results pertaining to the
CNTFR
The six
residues identified in the functional
The location of the
To directly test the effect of the D30Q mutation on gp130 binding,
we followed the formation of bi- and tripartite complexes by native
polyacrylamide gel electrophoresis. Fig. 5shows that when a
small molar excess of human CNTF was mixed with sCNTFR
A common receptor epitope
topography between CNTF and GH would implicate a common topography for
their respective receptor complexes, particularly since both receptors
appear to consist of fibronectin domains that are predicted to be
involved in cytokine binding
(15) . Indeed, computer modeling of
the two carboxyl-terminal fibronectin domains of CNTFR
To further
investigate this common topography, we substituted the structure of
CNTF for GH in the crystal structure of its complex with the high
(
From mutational analysis of IL-6, the
specificity-conferring IL-6R
Based on the crystal structure of LIF and information from
domain exchange mutagenesis
(23, 24) , it was proposed
that LIFR and gp130 bind LIF at sites that only in part correspond to
the two receptor epitopes of GH
(25) . LIFR was predicted to bind
at a site analogous to the first GH epitope and at a second site
consisting of parts of the BC and CD loops and the beginning of helix
D. Furthermore, it was suggested that LIFR interacts with CNTF in the
same manner, and, as a consequence, a fourth, hypothetical site at an
unknown location would be required for CNTFR
In CNTF, the
locations of the first two epitopes and the crystal structure show that
the BC and CD loops and the beginning of helix D lie in an area that
would be uniformly accessible by a third receptor component such as
LIFR. This area, designated here epitope III, is characterized, as in
LIF, by the kink at the center of helix D
(13) . Thus, the second
proposed site for LIFR binding on LIF is also available for LIFR
binding on CNTF. Therefore, it appears very likely that epitope III is
occupied by LIFR in both LIF and CNTF.
This assignment expands the
receptor epitope spatial conservation to a third element. By analogy to
CNTF and LIF, a third epitope at an equivalent site would also be
located on the surface of IL-6 for the recognition of the second gp130
molecule. Thus, in long-chain helical cytokines interacting with a
third receptor component, a third receptor epitope involving residues
contributed by the BC and CD loops and the beginning of helix D appears
also to be conserved. Other long chain helical cytokines, such as IL-11
and oncostatin M, that use gp130 and/or LIFR for signal transduction
would also be expected to maintain the same receptor epitope topology.
In fact, it would be interesting to see whether similar patterns emerge
from other families of structurally related proteins, such as the short
chain helical cytokines or even non-helical proteins. In this respect,
the conservation of a common epitope order and its relation to
functional specificity may be related to parallel evolution mechanisms
of cytokines and their receptors.
Potency values are expressed relative to human CNTF. Complex
formation with sCNTFR
Values are as in the legend to Table I.
ND, values not determined.
and
not with the
-components, gp130 and LIFR. In the crystal
structure, the side chains of these residues, which are located in
helix A, the AB loop, helix B, and helix D, are surface accessible and
are clustered in space, thus constituting an epitope for CNTFR
. By
the same analysis, a partial epitope for gp130 was also identified on
the surface of helix A that faces away from the
-epitope.
Superposition of the CNTF and growth hormone structures showed that the
location of these epitopes on CNTF is analogous to the location of the
first and second receptor epitopes on the surface of growth hormone.
Further comparison with proposed binding sites for
- and
-receptors on interleukin-6 and leukemia inhibitory factor
indicated that this epitope topology is conserved among helical
cytokines. In each case, epitope I is utilized by the
specificity-conferring component, whereas epitopes II and III are used
by accessory components. Thus, in addition to a common fold, helical
cytokines share a conserved order of receptor epitopes that is function
related.
(
)
displays trophic activity on a variety of neuronal cells
both in vivo and in vitro, prevents the degeneration
of motor neurons after axotomy, and has myotrophic activity on
denervated skeletal muscle (reviewed in Refs. 1 and 2). The biological
activity of CNTF is transduced by a multi-component receptor system
that consists of the primary ligand binding
-component
(CNTFR
) and two transmembrane
-components, gp130 and LIFR.
One or both of the latter are shared components of the receptors for
leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and several
other cytokines
(3) . Signal transduction ensues through the
interaction of these receptor components with the Jak-Tyk family of
cytoplasmic protein kinases and leads to the tyrosine phosphorylation
of p91, the related acute phase response factor and other proteins that
are also phosphorylated in response to various cytokines and growth
factors
(4, 5, 6) .
is anchored to
the cell surface through a GPI link but is also found in released form
at increased levels following nerve injury
(7, 8) . In
``soluble'' (sCNTFR
) form, recombinant receptor can
confer survival to normally non-responsive, non-neuronal TF-1 cells
that lack CNTFR
by reconstituting active receptor complexes with
the
-components on the cell surface
(9, 10) . Added
sCNTFR
can also elicit a proliferative response to mouse BAF-B03
cells coexpressing recombinant LIFR plus gp130
(11) .
potentiates the activity of different CNTF variants on TF-1
cells in a quantitative order that reflects the affinity of the
cell-bound CNTFR
for these ligands
(12) . Thus, sCNTFR
reconstitutes on the cell surface active complexes that are analogous
to the cell-attached form. CNTF and sCNTFR
form a stable 1:1
complex, whose size indicates that the two proteins interact as
monomers
(12) . Dynamic light-scattering experiments show that
human CNTF is monomeric at concentrations up to approximately 45
µM, i.e. 1 mg
ml
,
although CNTF becomes primarily dimeric at higher concentrations.
Indeed, the x-ray structure of crystals obtained at 10
mg
ml
shows a specific dimer in a head-to-tail
arrangement, which may represent a storage form of CNTF
(13) .
The structure also shows that CNTF is folded in the four-helix bundle
arrangement exhibited by many cytokines, although two of the helices, A
and D, are kinked
(13) .
, cross-linking experiments suggest
that CNTF makes direct contact with the other components of the
receptor complex on the cell surface. Indeed, rat CNTF has been
cross-linked to transfected human CNTFR
on the surface of cell
lines
(7) and to both endogenous and transfected gp130 and
LIFR
(14) . However, binding of rat CNTF in the absence of the
-component to cells expressing recombinant gp130 or LIFR could not
be detected, even though rat CNTF bound with high affinity to mouse
BAF-B03 cells coexpressing recombinant human CNTFR
plus LIFR and
with lower affinity to cells coexpressing CNTFR
plus
gp130
(11) .
(I) and a second, distinct site
(II) that is involved in binding gp130. Comparisons with growth hormone
(GH), IL-6, and LIF show that epitope I is preserved for binding the
primary, specificity-determining component, whereas epitope II (and III
in the case of CNTF, IL-6, and LIF in part) is preserved for
recognition of accessory receptors.
Genetic Engineering, Protein Purification, and
Characterization
All proteins were engineered, extracted,
refolded, and purified to at least 90% purity and characterized with
the methods previously described
(12, 16, 17) except for the extracellular domain of gp130, which was
purchased (R& Systems). Purity and structural integrity were
assessed on reducing 15% SDS-polyacrylamide gels. A few key human CNTF
variants such as Q63R and Q63R,W64A were further purified by
reverse-phase chromatography to better than 99% purity and re-assayed
with identical results. Conformational purity was assessed by native
gel electrophoresis, whereby all proteins showed the presence of one or
a few major discrete bands. Native gel electrophoresis was also used to
monitor binding to sCNTFR
(12) . Complex formation with
sCNTFR
was monitored on native gels according to the legend of
Fig. 5
. Without exception, binding to sCNTFR
was clearly
evident with CNTF variants that responded normally to sCNTFR
in
the TF-1 assay but was not detectable with mutants that showed weak
functional interaction with sCNTFR
.
Figure 5:
Multiple receptor complex formation
monitored on native gels. Human CNTF or the Asp-30 variant (4 µg
each) were mixed with 3 µg of sCNTFR plus 0, 1, or 2 µg of
gp130 in 20 µl total volume and analyzed on 7% native
polyacrylamide gels as described (12).
Biological Activity Assays
Biological
activity was measured on dissociated E8 chicken ciliary ganglion
neurons and on TF-1 human erythroleukemia cells as
described
(12) . Potency values were expressed relative to human
CNTF. Potency was defined as the reciprocal ligand concentration
showing the same biological activity as 1 ng/ml human CNTF for the
ciliary neuron assays or 100 ng/ml human CNTF for the TF-1 cell assays.
These values were obtained from concentration response curves of the
type shown in Fig. 1by graphic extrapolation. Potentiation by
sCNTFR was measured from the increase in potency caused by the
addition of 100 ng/ml sCNTFR
relative to the increase caused by
the same amount of receptor on human CNTF. For each protein, at least
two independent concentration response curves were obtained. Potency
values obtained from such curves varied by less than 2-fold, which
represents the error limits.
Figure 1:
Functional assays specific for the
complete 1
2 receptor complex (neurons), the
1
2
receptors (TF-1), or soluble
receptor (TF-1 cells +
sCNTFR
) for human CNTF and variants Q63E and D30Q. Human CNTF
(
), human CNTF variants (
), and human CNTF variants in the
presence of 20 (
) or 100 ng/ml (
) sCNTFR
are
shown.
Functional Evaluation of CNTF
Variants
To distinguish the interaction of CNTF with the
various components of its receptor, we used three quantitative cell
survival assays. The first measures activity mediated by the complete
receptor complex present in primary neurons; the second measures
activity mediated by the -components on F-1 cells; and the third
measures interaction with the
-component added as sCNTFR
on
TF-1 cells
(12) .
Wild Type Receptor Response
A variety of
effects on the biological activity of CNTF was observed as a
consequence of individual amino acid substitution at various positions
within human CNTF, for example the substitution D30Q (Fig. 1).
However, substitution of different amino acids at the same position had
profound effects on the survival of primary neurons in vitro that varied from nearly complete loss to a strong gain of activity
(Fig. 1; Tables I and II). For example, the substitution Q63E
caused a greater than 100-fold loss of potency on ciliary neurons,
whereas substitution of an arginine at this position has been shown to
increase the potency of human CNTF 4-5-fold
(12) . Because
ciliary neurons possess the complete receptor complex, these effects
could arise from an altered interaction with the - or
-receptor components.
Different results
were obtained when the potency of these proteins was determined on TF-1
cells, which respond through a residual affinity of CNTF for the
gp130/LIFR components
(12) . The Q63E mutant, which was nearly
inactive on primary neurons, was as potent as human CNTF in promoting
the survival of TF-1 cells (Fig. 1). The full potency of the Q63E
mutant toward TF-1 cells indicates that the side chain of this amino
acid is not involved in the interaction of the CNTF molecule with the
-Receptor Response
-receptor components. Therefore, the greatly decreased activity of
this mutant on primary neurons must result from impaired interaction
with the
-receptor alone. Fig. 1also shows that the D30Q
mutant is approximately 10 times less active than human CNTF on TF-1
cells, a result indicating that the side chain at position 30 is
important for the interaction of CNTF with one or both of the
-receptor components. The fact that the relative potency of this
protein on primary neurons parallels its relative potency toward TF-1
cells is consistent with this interpretation because weaker functional
interaction with a
-receptor should impair biological activity in
both assays. Overall, the relative potency of the various mutants
toward TF-1 cells and primary neurons allows functional dissection of
the interaction of CNTF with the
- and the
-receptor
components; the results indicate that distinct parts of the protein
contribute to these interactions.
Response to sCNTFR
To obtain
biologically relevant evidence that the reduced activity of the Q63E
protein on primary neurons was indeed due to a weaker functional
interaction with CNTFR, we took advantage of the response of TF-1
cells to sCNTFR
. As previously shown, added receptor potentiates
the response of TF-1 cells to CNTF in a manner that reflects the
relative affinity and specificity of the cell-attached form of the
receptor
(12) . Addition of 100 ng/ml sCNTFR
on the dose
response of Q63E causes a less than 2-fold shift (Fig. 1). This
value is only 16% of the shift observed for wild type CNTF
(12) ,
indicating very weak formation of functional complexes with sCNTFR
(). This result is consistent with both the lower
biological activity of Q63E on primary neurons and its normal activity
on TF-1 cells and provides direct evidence that this modification
affects functional interaction with CNTFR
but not with the
-receptors.
caused a shift in the concentration
response of D30Q, comparable to that of human CNTF, even though this
ligand is much less active than human CNTF in the absence of added
receptor ( Fig. 1and ). This result indicated that
the D30Q variant interacts normally with sCNTFR
. Therefore, the
lower potency of D30Q on primary neurons and TF-1 cells reflects
impaired interaction with the
-component(s) and indicates that
position 30 of the CNTF molecule is involved in
-component
recognition.
-type, consists of proteins
characterized by lower activity on primary neurons, normal activity on
TF-1 cells, and little potentiation by sCNTFR
. These proteins
carry mutations that impair their functional interaction with
sCNTFR
but not with a
-receptor component, as exemplified by
Q63E. The second,
-type, consists of proteins characterized by
lower activity on primary neurons, lower activity on TF-1 cells, but
normal potentiation by sCNTFR
. These mutants carry structural
changes that impair the interaction of CNTF with one or both of the
-receptor components but not with CNTFR
, as exemplified by
D30Q. By applying this functional analysis to CNTF variants carrying
side chain substitutions at different positions of the molecule, we
identified proteins whose properties classified them into one or the
other of these types.
A Surface Epitope for CNTFR
shows that side chain substitutions at amino acid residues
Arg-25, Arg-28, Gln-63, Trp-64, Gln-74, Asp-175, and Arg-177 of human
CNTF drastically affect its functional interaction with CNTFR but
have a minimal or no effect on activity mediated by the
-receptor
components. This property indicates that only the part of the CNTF
molecule involved in CNTFR
recognition is affected by these
mutational changes but not the overall protein conformation or
stability. Therefore, the amino acid side chains at these positions are
likely to constitute a binding epitope for CNTFR
, although
localized conformational changes around these residues could not be
ruled out.
. Most importantly, however, these residues are
grouped in three-dimensional space (Fig. 2), even though they are
located distantly along the primary protein sequence. Residues 25 and
28 are located in helix A, residues 63 and 64 are located in the AB
loop, residue 74 is located in helix B, and residue 177 is located in
helix D (Fig. 3). The surface accessibility of these residues and
their clustering in space strongly supports the conclusion that they
define an epitope for CNTFR
. The evidence also indicates that the
side chains of these residues are directly involved in receptor
contact. However, the possibility of additional localized
conformational changes cannot be ruled out, particularly for residues
63 and 64 in the flexible AB loop.
Figure 2:
An
electrostatic potential surface of the CNTF monomer. Blue,
positive; red, negative potential. Superposed onto the surface
are the six residues in the CNTFR epitope. For the disordered
portions of the AB and CD loops not resolved in the crystal structure
(13), approximate positions of the main chain atoms were modeled and
included into the surface calculation. This figure was generated with
the program GRASP (26).
Figure 3:
Alignment of CNTF, GH, and IL-6. CNTF was
aligned to GH by superposition of their crystal structures and to IL-6
by superposition to the structure of G-CSF and sequence alignment of
G-CSF and IL-6, as described (13). Residues conserved in a visual
alignment of the primary sequences of human, rabbit, and rat CNTF, as
well as chicken growth-promoting activity (27) are also shown
(Cons). Residues in the first and secondreceptorepitope of each molecule are colored
green and magenta, respectively. Residues in the four
helices are boxed. For GH, residues having more than a 20
Å decrease in solvent accessibility upon receptor
binding were included in each receptor epitope (19). For IL-6, the
residues assigned to the epitope for gp130 are from Ref. 20. CNTF
residues whose substitution by alanine has no measurable effect on
biological activity are colored
blue.
One exception to the surface
accessibility of the side chains of mutants that are impaired in their
interaction with CNTFR is D175A. The x-ray structure shows that
the side chain of residue 175 is not surface accessible despite its
negative charge and, therefore, cannot be part of the CNTFR
binding epitope. This apparent discrepancy between the functional and
structural analysis of this mutant was reconciled when it was also
found that, in one of the two conformations of the AB loop in the
crystal structure, the side chain of Asp-175 forms hydrogen bonds with
residues, Trp-64, Arg-72, Asn-76, and Arg-171. Of these, Trp-64 is one
of the residues of the CNTFR
binding epitope. Therefore,
disruption of the hydrogen bond that connects Asp-175 to Trp-64 most
likely destabilizes the orientation of Trp-64 and possibly other
residues in the AB loop and perturbs the conformation of the CNTFR
binding epitope.
(
)
and hybrid
molecules
(16, 17) that have been generated thus far,
only one other substitution at a different position, A117R, partially
conveyed
-type characteristics. This rather drastic substitution
was designed so as to disrupt dimer formation, because in the crystal
structure Ala-117 is buried in the dimer interface. Indeed, A117R does
not form dimers and has a tendency to aggregate, which complicates the
analysis of its properties.
(
)
Numerous other
substitutions had no measurable effect on potency, implying weak or no
involvement with receptor binding, and a few others showed general loss
of activity, most likely due to global structural changes (
and Fig. 3). The rationale for these substitutions
was the early identification of the limits of amino and COOH terminus
deletion and the importance of the AB loop for receptor
binding
(16, 17) . Also, the prediction that by analogy
to GH, helix A, the AB loop, and residues 167-170 in helix D
should define a receptor epitope
(15) . However, among the
alanine variants that showed no measurable loss of activity were
residues 167-169.
epitope, Arg-25, Arg-28, and Arg-177, constitute a strong positively
charged cluster (Fig. 2). The fact that the substitution Q63R,
which adds a fourth positive charge to this cluster, creates a protein
with greatly enhanced potency and affinity for CNTFR
suggests the
importance of electrostatic interactions. However, a lysine side chain
substitution, which maintains the positive charge at this position, has
the opposite effect, causing selective loss of response to sCNTFR
and maintains the properties of an
-type variant ().
Furthermore, the substitution R177A, which removes a positive charge,
causes a relatively small decrease in potency. Therefore, the nature of
the side chains appears to be far more important. The role of the
positively charged cluster in the
-epitope may be to accelerate
receptor docking through electrostatic attraction, as proposed for
GH
(18) .
epitope, the human CNTF amino acid residues at positions 25,
28, 63, 64, 74, and 177 were shown to constitute a functional epitope
because they meet the following criteria. First, mutations at those
positions specifically disrupt functional interaction with CNTFR
but not with the
-receptor components (). Second, the
side chains of these residues are exposed on the surface of the CNTF
molecule and are found to be clustered together so that they would be
uniformly accessible for receptor recognition (Fig. 2). Third,
these residues are conserved across species (Fig. 3) with the
exception of the glutamine residues at positions 63 and 74, which are
arginine and leucine in chicken growth-promoting activity
(27) ,
respectively; gln-63 is an arginine in rat CNTF as well.
-epitope on CNTF most likely
constitute the majority of the functional receptor contacts. In the
GH-receptor I complex, 31 side chains are buried in the interface, but
the functional epitope consists of a quarter of these residues that are
clustered near the center of the structural epitope and can account for
the majority of the binding energy
(18) .
-binding epitope on the surface of CNTF is reminiscent of the
first receptor binding epitope on the human GH molecule
(19) ,
which consists of amino acids contributed by helix A, the AB loop, and
helix D. Three-dimensional superposition of the C
atoms of CNTF to
those of GH indicated that the two epitopes are at equivalent positions
along helices A and D and the AB loop
(13) . In fact, five out of
the six CNTF residues in the
-receptor epitope, 25, 28, 63, 64,
and 177, have counterparts in the first receptor epitope of GH
(Fig. 3). Thus, the respective locations of the primary receptor
epitopes on the surface of GH and CNTF are equivalent, even though the
two proteins are not related by primary sequence and interact with
different numbers of unrelated receptors.
A Partial
also summarizes the properties of CNTF
mutants characterized by functionally altered interactions with a
-Receptor Epitope for
gp130
-receptor component. Only one side chain substitution, D30Q,
clearly results in a molecule specifically impaired in its interaction
with a
-receptor component but not with CNTFR
, i.e. lower activity on primary neurons and TF-1 cells but normal
potentiation by sCNTFR
. A second substitution, K26A, also caused
reduced potency toward primary neurons, but, surprisingly, it also
caused a 6-fold greater potency toward TF-1 cells and reduced
potentiation by sCNTFR
. Thus, the K26A substitution does not
result in a clear
-type. Remarkably, the crystal structure of
human CNTF shows that the side chains of residues 26 and 30 in helix A
are surface accessible, interact via a salt bridge, and are positioned
(13) away from the face of helix A, which contributes to
-receptor binding (Fig. 4B). Furthermore,
superposition of the C
atoms of CNTF and human GH shows that the
C
atom of the Lys-26 is superposable on the C
atom of GH
Arg-19 (Fig. 3), which is one of the residues in the second
receptor binding epitope of GH
(19) . These results indicate that
residues 26 and 30 of CNTF partially define an epitope for a
-component at a solvent-accessible site that is analogous to the
second receptor epitope of GH.
Figure 4:
Speculative model for the interaction of
CNTF with two of its receptor subunits, based on the similarity of the
CNTF and GH structures and the locations of their receptor epitopes.
This model was derived by superimposing the structure of CNTF
(blue) onto that of GH in its complex with the high (,
green) and the low (
1, magenta) affinity
receptors. CNTF residues in the epitope for CNTFR
are facing the
-receptor, whereas those in the gp130 epitope are oriented toward
the
-receptor. B, close-up view. Alternatespheres represent the poorly ordered portions of the AB
and CD loops in the crystal structure (13). Figures were generated with
the program GRASP (26).
To obtain further insight as to the
nature of the -component that interacts with this epitope, the
sequence of CNTF was first aligned with that of G-CSF by superposition
of their C
atom frameworks and then aligned to human IL-6 using
the significant identity between the latter two sequences as
described
(13) . The alignment between G-CSF and IL-6 obtained by
this approach agrees with that obtained by computer
modeling
(20) . Fig. 3shows that residues Lys-26 and
Asp-30 of human CNTF correspond to residues Tyr-31 and Gly-35 of human
IL-6, which have been reported to affect binding of IL-6 to
gp130
(20) . This result indicated that residues 26 and 30
partially define on the surface of CNTF an epitope (
1) for gp130.
, the
majority of both proteins formed a bipartite complex that migrated to a
new position of the gel that corresponds to the CNTF
sCNTFR
complex, as previously shown
(12) . Upon addition of 1 µg
(
1/4 molar equivalent) of gp130, part of the bipartite complex
shifted to a new position of the gel. Addition of 2 µg of gp130
further titrated the ligand
sCNTFR
complex to the position of
the tripartite complex. At the same time, the intensity of the free
ligand band did not change significantly, indicating that gp130 has
greater affinity for the ligand
sCNTFR
complex than for free
ligand (and for free sCNTFR
, as shown in other control
experiments). A quantitatively different result was obtained with the
D30Q variant. Addition of 1 µg of gp130 to the
D30Q
sCNTFR
complex did not titrate a significant amount of
complex; only upon addition of 2 µg of gp130 some tripartite
complex formation was observed (Fig. 5). Therefore, the affinity
of the D30Q
sCNTFR
complex for gp130 is weaker that the
affinity of the human CNTF
sCNTFR
complex, consistent with
the conclusion that Asp-30 partially defines a gp130 epitope. This
analysis provides the first direct evidence for tripartite complex
formation in solution and is consistent with the results of competitive
binding experiments, showing that gp130 on cell surfaces has no
measurable affinity for CNTF in the absence of
-receptor
(11) . By this method, evidence of an
IL-6
sIL-6R
gp130 complex was also obtained.
(
)
A Common Function-related Spatial
Arrangement of Receptor Epitopes among Helical Cytokines
By
combining the information obtained from mutational analysis and the
crystal structure, we identified on CNTF two independent receptor
epitopes for CNTFR and gp130. Superposition of the CNTF structure
on that of GH revealed that the two epitopes in CNTF are located at
positions topologically analogous to the first and second receptor
epitopes of GH. This finding is remarkable, in view of the fact that
the two proteins are not related by primary sequence or function and
interact with different numbers of unrelated receptors. GH forms a
complex with two molecules of the same receptor, whereas, CNTF forms a
complex with three different receptors. Yet, the topography of the
epitopes for the first two receptors that bind each ligand is
maintained. Furthermore, in both molecules, epitope I is involved in
the recognition of the first, specificity-conferring ligand, whereas
epitope II is involved in binding a second accessory receptor.
Mechanistically, the latter is also the second receptor to bind
sequentially. This unusual conservation of receptor epitope topography
points to a mode of receptor component engagement that would be common
to long chain helical cytokines.
showed that
its conformation is compatible with the crystal structure of the GHR
without any apparent main chain clashes.
) and the low (
1) affinity receptors. Remarkably, we
observed that all residues in the epitope for CNTFR
were facing
the high affinity growth hormone receptor GHR, whereas the two residues
in the gp130 epitope were oriented toward the low affinity GHR
(Fig. 4A). In fact, five of the six residues would be
capable of direct receptor contact if the CNTFR
occupied the
position of the first GH receptor molecule in the crystal structure of
the tripartite complex (Fig. 4B). It is conceivable that
the sixth residue, Gln-74, which is located further away from the
surface, defined by the other residues might make contact with the
third fibronectin type III domain of CNTFR
, which is not present
in the GHR.
was proposed to interact with two
residues that a computer model placed at the
COO
-terminal of helix
D
(15, 20, 21, 22) . Also, the
interaction of the first gp130 receptor molecule with IL-6 is impaired
by the simultaneous modification of two residues similarly assigned in
helix A
(20) . Even though the structure of IL-6 is not known,
the alignment shown in Fig. 3agrees with the assignment of these
residues at locations topologically analogous to epitopes I and II of
CNTF, respectively. Thus, to a first approximation the epitope
topology, conservation between GH and CNTF appears to apply to IL-6, as
well.
binding
(25) .
The evidence presented here disagrees with the speculation regarding
the binding site for CNTFR
but is otherwise consistent with the
proposed topology of receptor epitopes on LIF. In fact, the proposal
that the first site of LIFR binding is at a location analogous to the
first GHR epitope is in agreement with our conclusion that epitope I is
conserved for the specificity-conferring component.
Table:
Distinct types of human CNTF variants
was monitored on native gels according to
the legend of Fig. 5.
Table:
Other amino acid
substitutions in human CNTF
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.