Human IL-21 and IL-4 bind to partially overlapping epitopes of common gamma-chain

Interleukin 21 (IL-21) is a recently identified novel cytokine that plays an important role in the regulation of B, T, and NK cell functions. Its effects depend on binding to and signaling through an IL-21 receptor complex consisting of the IL-21 receptor (IL-21R) and the common gamma-chain (gamma(c)). In this study using biosensor technique, the ligand-binding properties of IL-21R and gamma(c), which are presently poorly understood on a molecular level, were analyzed employing recombinant ectodomains of IL-21R and gamma(c). The formation of a binary complex between IL-21 and immobilized IL-21R (K(D) 70pM), gamma(c) and immobilized IL-21 (K(D) 160 microM) and a ternary complex between gamma(c) and IL-21 saturated immobilized IL-21R (K(D) 160nM) could be analyzed. The gamma(c) residues involved in IL-21 binding were defined by alanine-scanning mutational analysis. The epitope comprises gamma(c) residues N44, Y103, N128, L161, E162, and L208. It is not identical but partially overlapping with the previously established gamma(c) epitope for IL-4 binding. These results open the way to understand the molecular recognition mechanism in the IL-21 receptor system and also the promiscuous binding properties of gamma(c).     

Molecular and structural basis of cytokine receptor pleiotropy in the interleukin-4/13 system

Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R alpha/gamma(c)/IL-4) and type II (IL-4R alpha/IL-13R alpha1/IL-4, IL-4R alpha/IL-13R alpha1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for gamma(c)'s ability to recognize six different gamma(c)-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R alpha1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.     

Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its alpha, beta, and gamma(c) receptors

Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the alpha, beta and the "common gamma" (gamma(c)) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor-ligand, and receptor-receptor interactions that are necessary for formation of the high-affinity IL-2/alphabetagamma(c) signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its alpha receptor, a moderately entropically favorable low affinity interaction between IL-2 and its beta receptor, and no interaction between IL-2 and the shared receptor, gamma(c). Formation of the stable intermediate trimolecular complexes of IL-2 with alpha and beta receptors, as well as IL-2 with beta and gamma(c) receptors proceeds through enthalpy-entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor alpha and beta chains, suggesting that a preformed alphabeta complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Ralphabetagamma(c) high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by gamma(c) contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.     

Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.

The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.     

The constraints protein-protein interactions place on sequence divergence

IL4-BP, the extracellular binding domain of the IL-4 receptor alpha chain, contains a high-affinity binding epitope for IL-4 (K(D) 150 pM). Previous results on the crystal structure of the IL-4/IL4-BP complex and on the functional epitope of IL-4 suggested that this contact comprises a mosaic of two binding clusters. The present mutational analysis of IL4-BP supports this view and demonstrates that the energetically most important group is the receptor carboxylate group of D72 forming an ion pair with IL-4 R88 in cluster II. The second main receptor determinant is the hydroxyl group of Y183 forming a hydrogen bond with IL-4 E9 in cluster I. The latter is engaged in additional hydrogen bonds with Y13 and also in van der Waals contacts with Y127. Receptor residue D72 as well as Y183 are each surrounded by a shell of hydrophobic groups from residues that upon mutation lead to smaller decreases in binding affinity. Analysis of IL4-BP double mutants showed that receptor side-chains within one cluster but not those of different clusters cooperate. Interaction analysis of IL-4 and IL4-BP single mutants also revealed additivity in binding of side-chains between clusters and cooperativity within each cluster I or II.These results show that the high-affinity IL-4/IL4-BP contact is constituted by two independent binding units, each containing a central polar or charged side-chain surrounded by hydrophobic groups (avocado cluster).     

Interleukin-4-specific signal transduction events are driven by homotypic interactions of the interleukin-4 receptor alpha subunit.

Interleukin-4 (IL-4) exerts its effects through a heterodimeric receptor complex (IL-4R), which contains the IL-4R(alpha) and gamma(c) subunits. IL-4R(alpha) also functions with other partner subunits in several receptor types, including the IL-13 receptor. To examine the roles of the individual subunits within IL-4R complexes, we employed a chimeric system that recapitulates native IL-4R function as verified by the activation of the kinases, JAK1 and JAK3, and induction of STAT-6. When a mutant gamma(c) subunit in which the four cytoplasmic tyrosines were converted to phenylalanine was paired with the cytoplasmic domain of the IL-4R(alpha) chain, specificity within the JAK-STAT pathway was not altered. Signaling events were examined further in cells expressing the IL-4R(alpha) chimera alone without the gamma(c) chimera. Ligand-induced homodimerization of these receptors activated the IL-4 signaling program despite the absence of gamma(c), including induction of JAK1 and STAT-6, phosphorylation of the insulin-related substrate 1 and cellular proliferation. Thus, homotypic interactions of the IL-4R(alpha) subunit are sufficient for the initiation and determination of IL-4-specific signaling events, and such interactions may be integral to signaling through IL-4R complexes.     

Crystal structure of the interleukin-4/receptor alpha chain complex reveals a mosaic binding interface

Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling. The crystal structure of the intermediate complex between human IL-4 and IL4-BP was determined at 2.3 A resolution. It reveals a novel spatial orientation of the two proteins, a small but unexpected conformational change in the receptor-bound IL-4, and an interface with three separate clusters of trans-interacting residues. Novel insights on ligand binding in the cytokine receptor family and a paradigm for receptors of IL-2, IL-7, IL-9, and IL-15, which all utilize gamma c, are provided.     

Characterization of the recombinant extracellular domains of human interleukin-20 receptors and their complexes with interleukin-19 and interleukin-20

The soluble extracellular domains of human interleukin-20 (IL-20) receptors I and II (sIL-20R1 and sIL20R2), along with their ligands IL-19 and IL-20, were expressed in Drosophila S2 cells and purified to homogeneity. Formation of the receptor/receptor and ligand/receptor complexes was studied by size exclusion chromatography. Both ligands and soluble receptors were found to be monomeric in solution; homo- or heterodimers are not formed even at elevated concentrations. Under native conditions, both IL-19 and IL-20 form stable ternary 1:1:1 complexes with the sIL-20R1 and sIL20R2 receptors, as well as high-affinity binary complexes with sIL-20R2. Unexpectedly, sIL-20R1 does not bind on its own to either IL-19 or IL-20. Thus, one of the possible consecutive mechanisms of formation of the signaling ternary complex may involve two steps: first, the ligand binds to receptor II, creating a high-affinity binding site for the receptor I, and only then does receptor I complete the complex.     

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2. STRUCTURED SUMMARY:     

Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay

Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Rα1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Rα1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Rα1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Rα1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100 nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Rα1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.     

Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the gamma chain

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between specific interleukin receptor subunits, which bind Janus kinase 1 (JAK1) and the JAK3 binding common gamma chain (gamma c). The potential existence and roles of homomeric and heteromeric complexes before ligand binding and their modulation by ligand and JAK3 are unclear. Using computerized antibody-mediated immunofluorescence co-patching of epitope-tagged receptors at the surface of live cells, we demonstrate that IL-2Rbeta, IL-9Ralpha, and gamma c each display a significant fraction of ligand-independent homomeric complexes (24-28% co-patching), whereas control co-patching levels with unrelated receptors are very low (7%). Heteromeric complex formation of IL2-Rbeta or IL-9Ralpha with gamma c is also observed in the absence of ligand (15-30%). Ligand binding increases this hetero-oligomerization 2-fold but does not affect homo-oligomerization. Co-expression of IL-2Ralpha does not affect the hetero-oligomerization of IL-2Rbeta and gamma c. Recruitment of gamma c into heterocomplexes is partly at the expense of its homo-oligomerization, suggesting that a functional role of the latter may be to keep the receptors inactive in the absence of ligand. At the same time, the preformed complexes between gamma c and IL-2Rbeta or IL-9Ralpha promote signaling by the JAK3 A572V mutant without ligand, supporting a pathophysiological role for the constitutive oligomerization in triggering ligand-independent activation of JAK3 (and perhaps other JAK mutants) mutants identified in several human cancers.     

A model of the ternary complex of interleukin-10 with its soluble receptors

Background  

Interleukin-10 (IL-10) is a cytokine whose main biological function is to suppress the immune response by induction of a signal(s) leading to inhibition of synthesis of a number of cytokines and their cellular receptors. Signal transduction is initiated upon formation of a ternary complex of IL-10 with two of its receptor chains, IL-10R1 and IL-10R2, expressed on the cell membrane. The affinity of IL-10R1 toward IL-10 is very high, which allowed determination of the crystal structure of IL-10 complexed with the extracellular/soluble domain of IL-10R1, while the affinity of IL-10R2 toward either IL-10 or IL-10/sIL-10R1 complex is quite low. This so far has prevented any attempts to obtain structural information about the ternary complex of IL-10 with its receptor chains.     

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.     

Crystal structure of a soluble decoy receptor IL-22BP bound to interleukin-22

Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75 Å resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.

Structured summary

MINT-7010533: IL-22 BP (uniprotkb:Q969J5) and IL-22 (uniprotkb:Q9GZX6) bind (MI:0407) by X-ray crystallography (MI:0114)     

The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3

The common cytokine receptor gamma chain (gamma c), an essential component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. Recently, a novel lymphokine (IL-21) and its receptor (IL-21R alpha) were described which profoundly affect the growth and activation state of B, T, and NK cells in concert with other lymphokines or stimuli [Parrish-Novak, J., et al. (2000) Nature 408, 57-63]. In this report, we show that gamma c is also a required signaling component of the IL-21 receptor (IL-21R) using the gamma c-deficient X-linked severe combined immunodeficiency (XSCID) lymphoblastoid cell line JT, and JT cells reconstituted with gamma c (JT/gamma c). Moreover, we demonstrate a functional requirement for both gamma c and the gamma c-associated Janus family tyrosine kinase 3 (JAK3) in IL-21-induced proliferation of pro-B-lymphoid cells engineered to express human IL-21R alpha (BaF3/IL-21R alpha). Retroviral-mediated transduction of wild-type gamma c into XSCID JT cells restored function to the IL-21R, as shown by IL-21-induced tyrosine phosphorylation of JAK1 and JAK3, and downstream activation of STAT5, in JT/gamma c cells as well as BaF3/IL-21R alpha and primary splenic B cells. In contrast, IL-21 failed to activate the JAK-STAT pathway in nonreconstituted JT cells. Monoclonal antibodies specific for the gamma c chain effectively inhibited IL-21-induced growth of BaF3/IL-21R alpha cells, supporting a functional role for this molecule in the IL-21R complex. In addition, the specific JAK3 tyrosine kinase inhibitor WHI-P131 significantly reduced IL-21-induced proliferation of BaF3/IL-21R alpha cells. Taken together, these results definitively demonstrate that IL-21-mediated signaling requires the gamma c chain, and indicate that JAK3 is an essential transducer of gamma c-dependent survival and/or mitogenic signals induced by this cytokine.     

Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes

The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed.     

A model of the complex between interleukin-4 and its receptors

A three-dimensional model of interleukin-4 (IL-4) bound to one molecule each of the high- and low-affinity receptors (IL-4R and IL-2Rγ) was built, using the crystal structure of the complex of human growth hormone (HGH) with its receptor (HGHR) as a starting model. The modeling of IL-4 with its receptors was based on the conservation of the sequences and on the predicted structural organization for cytokine receptors, and assuming that the binding mode of the ligands would be similar. Analysis of the interface between IL-4 and both receptor molecules was carried out to reveal which residues are important for complex formation. The modeling procedures showed that there were no major problems in maintaining a reasonable fit of IL-4 with the two receptor molecules, in a manner analogous to the complex of HGH–HGHR. Many of the residues that appear by modeling to be important for binding between IL-4 and the receptors have been previously implicated in that role by different methods. A striking motif of aromatic and positively charged residues on the surface of the C-terminal domains of the receptors is highly conserved in the structure of HGH–HGHR and in the models of IL-4 complexed with its receptors. © 1995 Wiley-Liss, Inc.     

Efficient production and characterization of Bacillus anthracis lethal factor and a novel inactive mutant rLFm-Y236F

The receptor binding to interleukin (IL)-13 is composed of the IL-13 receptor α1 chain (IL-13Rα1) and the IL-4 receptor α chain (IL-4Rα). In order to investigate the interaction of IL-13 with IL-13Rα1 and IL-4Rα, the DNA fragments coding the extracellular regions of human IL-13Rα1 and the IL-4Rα (containing a cytokine receptor homologous region) were fused with mouse Fc and expressed by a silkworm–baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. After further purification with anion-exchange chromatography, these receptors were used to investigate the ligand–receptor interaction. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13Rα1 showed predominant affinity to IL-4Rα, although neither detectable affinity of IL-13 nor IL-13Rα1 was observed against IL-4Rα. Combining these data with the moderate affinity of IL-13 to IL-13Rα1, this indicates that IL-13 first binds to IL-13Rα1 and recruits consequently to IL-4R.     

Functional epitope of common gamma chain for interleukin-4 binding.

Interleukin 4 (IL-4) can act on target cells through an IL-4 receptor complex consisting of the IL-4 receptor alpha chain and the common gamma chain (gamma(c)). An IL-4 epitope for gamma(c) binding has previously been identified. In this study, the gamma(c) residues involved in IL-4 binding were defined by alanine-scanning mutational analysis. The epitope comprises gamma(c) residues I100, L102, and Y103 on loop EF1 together with L208 on loop FG2 as the major binding determinants. These predominantly hydrophobic determinants interact with the hydrophobic IL-4 epitope composed of residues I11, N15, and Y124. Double-mutant cycle analysis revealed co-operative interaction between gamma(c) and IL-4 side chains. Several gamma(c) residues involved in IL-4 binding have been previously shown to be mutated in X-linked severe combined immunodeficiency. The importance of these binding residues for gamma(c) function is discussed. These results provide a basis for elucidating the molecular recognition mechanism in the IL-4 receptor system and a paradigm for other gamma(c)-dependent cytokine receptor systems.