©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Localization of Functional Receptor Epitopes on the Structure of Ciliary Neurotrophic Factor Indicates a Conserved, Function-related Epitope Topography among Helical Cytokines (*)

Nikos Panayotatos (1)(§), Elzbieta Radziejewska (1), Ann Acheson (1), Robert Somogyi (1), Anu Thadani (1), Wayne A. Hendrickson (2), Neil Q. McDonald (2)(¶)

From the (1) From REGENERON Pharmaceuticals Inc., Tarrytown, New York 10591-6707 and the (2) Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, College of Physicians and Surgeons of Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Ciliary neurotrophic factor (CNTF)() 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) .

CNTFR 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) .

Soluble CNTFR 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 mgml, although CNTF becomes primarily dimeric at higher concentrations. Indeed, the x-ray structure of crystals obtained at 10 mgml 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) .

In addition to the well characterized interaction of CNTF with CNTFR, 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) .

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 (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.


EXPERIMENTAL PROCEDURES

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 12 receptor complex (neurons), the 12 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.




RESULTS AND DISCUSSION

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.

-Receptor Response

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 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.

A different result was obtained with the D30Q protein. Addition of sCNTFR 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.

Overall, two distinct types of CNTF variants emerged from this analysis. The first, -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.

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. 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.

It is worth noting that among the more than 90 other amino acid substitution and deletion mutants ()() 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.

Three of the amino acids in the CNTFR 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) .

To summarize the results pertaining to the CNTFR 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.

The six residues identified in the functional -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) .

The location of the -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 -Receptor Epitope for gp130

also summarizes the properties of CNTF mutants characterized by functionally altered interactions with a -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.

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, the majority of both proteins formed a bipartite complex that migrated to a new position of the gel that corresponds to the CNTFsCNTFR 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 ligandsCNTFR 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 ligandsCNTFR 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 D30QsCNTFR 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 D30QsCNTFR complex for gp130 is weaker that the affinity of the human CNTFsCNTFR 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-6sIL-6Rgp130 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.

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 showed that its conformation is compatible with the crystal structure of the GHR without any apparent main chain clashes.

To further investigate this common topography, we substituted the structure of CNTF for GH in the crystal structure of its complex with the high () 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.

From mutational analysis of IL-6, the specificity-conferring IL-6R 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.

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 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.

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.

  
Table: Distinct types of human CNTF variants

Potency values are expressed relative to human CNTF. Complex formation with sCNTFR was monitored on native gels according to the legend of Fig. 5.


  
Table: Other amino acid substitutions in human CNTF

Values are as in the legend to Table I. ND, values not determined.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

Present address: Imperial Cancer Research Fund Unit for Structural Biology, Dept. of Crystallography, Birkbeck College, London WC1E 7HX, UK.

The abbreviations used are: CNTF, ciliary neurotrophic factor; GH, growth hormone; LIF, leukemia inhibitory factor; IL-6, interleukin-6; GHR, growth hormone receptor; G-CSF, granulocyte colony-stimulating factor.

N. Panayotatos, E. Radziejewska, and A. Acheson, unpublished data.

N. Panayotatos, unpublished results.

N. McDonald, unpublished results.


REFERENCES
  1. Acheson, A., and Lindsay, R. M.(1994) Semin. Neurosci. 6, 507-515
  2. Lindsay, R. M., Wiegand, S. J., Altar, C. A., and DiStefano, P. S. (1994) Trends Neurosci. 17, 182-190 [CrossRef][Medline] [Order article via Infotrieve]
  3. Davis, S., and Yancopoulos, G. D.(1994) Curr. Opin. Neurobiol. 3, 20-24
  4. Bonni, A., Frank, D. A., Schindler, C., and Greenberg, M. E.(1993) Science 262, 1575-1579 [Medline] [Order article via Infotrieve]
  5. Stahl, N., Boulton, T. G., Farruggella, T., Ip, N., Davis, S., Witthuhn, B., Quelle, F. W., Silvennoinen, O., Barbieri, G., Pellegrini, S., Ihle, J. N., and Yancopoulos, G. D.(1994) Science 263, 92-95 [Medline] [Order article via Infotrieve]
  6. Boulton, T. G., Stahl, N., and Yancopoulos, G. D.(1994) J. Biol. Chem. 269, 11648-11655 [Abstract/Free Full Text]
  7. Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V., Furth, M. E., Squinto, S. P., and Yancopoulos, G. D.(1991) Science 253, 59-63 [Medline] [Order article via Infotrieve]
  8. Ip, N. Y., McClain, J., Barrezueta, N., Aldrich, T. H., Pan, L., Li, Y., Wiegand, S., Friedman, B., Davis, S., and Yancopoulos, G. D.(1993) Neuron 10, 89-102 [Medline] [Order article via Infotrieve]
  9. Davis, S., Aldrich, T. H., Ip, N., Stahl, N., Scherer, S., Farruggella, T., DiStefano, P., Curtis, R., Panayotatos, N., Gascan, H., Chevalier, S., and Yancopoulos, G. D.(1993) Science 259, 1736-1739 [Medline] [Order article via Infotrieve]
  10. Davis, S., Aldrich, T. H., Stahl, N., Pan, L., Taga, T., Kishimoto, T., Ip, N. Y., and Yancopoulos, G. D.(1993) Science 260, 1805-1808 [Medline] [Order article via Infotrieve]
  11. Gearing, D. P., Ziegler, S. F., Comeau, M. R., Friend, D., Thoma, B., Cosman, D., Park, L., and Mosley, B.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1119-1123 [Abstract]
  12. Panayotatos, N., Everdeen, D., Liten, A., Somogyi, R., and Acheson, A. (1994) Biochemistry 33, 5813-5818 [Medline] [Order article via Infotrieve]
  13. McDonald, N. Q. Panayotatos, N., and Hendrickson, W. A.(1995) EMBO J., 14, in press
  14. Stahl, N., Davis, S., Wong, V., Taga, T., Kishimoto, T., Ip, N. Y., and Yancopoulos, G. D.(1993) J. Biol. Chem. 268, 7628-7631 [Abstract/Free Full Text]
  15. Bazan, J. F.(1991) Neuron 2, 197-208
  16. Masiakowski, P., Liu, H., Radziejewski, C., Lottspeich, F., Oberthuer, W., Wong, V., Lindsay, R. M., Furth, M. D., and Panayotatos, N. P. (1991) J. Neurochem. 57, 1003-1012 [Medline] [Order article via Infotrieve]
  17. Panayotatos, N., Radziejewska, E., Acheson, A., Pearsall, D., Thadani, A., and Wong, V.(1993) J. Biol. Chem. 268, 19000-19003 [Abstract/Free Full Text]
  18. Cunningham, B. C., and Wells, J. A.(1993) J. Mol. Biol. 234, 554-563 [CrossRef][Medline] [Order article via Infotrieve]
  19. De Vos, A. M., Ultsch, M., and Kossiakoff, A. A.(1992) Science 255, 306-312 [Medline] [Order article via Infotrieve]
  20. Savino, R., Lahm, A., Salvati, A. L., Ciapponi, L., Sporeno, E., Altamura, S., Paonessa, G., Toniatti, C., and Ciliberto, G.(1994) EMBO J. 13, 1357-1367 [Abstract]
  21. Lütticken, C., Kruttgen, A., Moller, C., Heinrich, P. C., and Rose-John, S.(1991) FEBS Lett. 282, 265-267 [CrossRef][Medline] [Order article via Infotrieve]
  22. Brakenhoff, J. P. J., de Hon, F. D., Fontaine, V., ten Boekel, E., Schooltink, H., Rose-John, S., Heinrich, P. C., Content, J., and Aarden, L. A.(1994) J. Biol. Chem. 269, 86-93 [Abstract/Free Full Text]
  23. Leebeek, F. W. G., Kariya, K., Schwabe, M., and Fowlkes, D. M.(1992) J. Biol. Chem. 267, 14832-14838 [Abstract/Free Full Text]
  24. Owczarek, C. M., Layton, M. J., Metcalf, D., Lock, P., Wilson, T. A., Gough, N. M., and Nicola, N.(1993) EMBO J. 12, 3487-3495 [Abstract]
  25. Robinson, R. C., Grey, L. M., Staunton, D., Vankelecom, H., Vernallis, A. B., Moreau, J.-F., Stuart, D. I., Heath, J. K., and Jones, E. Y. (1994) Cell 77, 1101-1116 [Medline] [Order article via Infotrieve]
  26. Nicholls, A., Sharp, K. A., and Honig, B.(1991) Proteins 11, 281-296 [Medline] [Order article via Infotrieve]
  27. Leung, D. W., Parent, A. S., Cachianes, G., Esch, F., Coulombe, J. N., Nikolics, K., Eckenstein, F. P., and Nishi, R.(1992) Neuron 8, 1045-1053 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.