ABSTRACT
Human interleukin-6 (hIL-6) is a pleiotropic cytokine with three distinct receptor epitopes, termed sites I, II, and III, which function to assemble a signaling complex. hIL-6 signals via a glycoprotein 130 (gp130) homodimer after initially forming a heterodimer with the nonsignaling α-receptor (IL-6Rα). The molecular description of the assembly of the hIL-6 signaling complex remains elusive because available structures provide descriptions of hIL-6 in its free and fully bound receptor forms, but not for intermediate steps that are crucial in the stepwise assembly of the signaling complex. In this report, molecular dynamics simulations provide atomic details describing the functional role of the initial hIL-6/IL-6Rα complex in facilitating subsequent interactions with gp130, which have not been previously shown. IL-6Rα binding to hIL-6 rigidifies the flexible N-terminus of the hIL-6 AB-loop through interactions with the D2 domain of IL-6Rα. This rigidification combined with repositioning of residues involved in gp130 receptor recognition promotes gp130 binding at site III. Binding of gp130 receptors at sites II and III is coupled with the release of the hIL-6 N-terminal AB-loop interaction and a pivoting of IL-6Rα around the hIL-6 helix bundle to the state of the hIL-6/IL-6Rα/gp130 complex.
Subject(s)
Interleukin-6 Receptor alpha Subunit/chemistry , Molecular Dynamics Simulation , Receptors, Interleukin-6/chemistry , Binding Sites , Dimerization , Glycoproteins/chemistry , Glycoproteins/metabolism , Humans , Interleukin-6 Receptor alpha Subunit/metabolism , Protein Binding , Protein Structure, Quaternary , Receptors, Interleukin-6/metabolism , ThermodynamicsABSTRACT
Interleukin (IL)-6 and IL-11 are the only canonical members of the IL-6 family of cytokines that induce signaling through a homodimer of the common ß-receptor glycoprotein (gp)130. A pre-requisite for signal transduction is the initial binding of the cytokines to their unique α-receptors, IL-6R and IL-11R. The cell-type specific expression of the two receptors determines the target cells of IL-6 and IL-11, because gp130 is ubiquitously expressed. However, ciliary neurotrophic factor (CNTF) and IL-27p28/IL-30 have been described as additional ligands for the IL-6R, underlining a remarkable plasticity among the cytokines of the IL-6 family and their receptors. In this study, we show that neither IL-6 nor IL-11 can bind to and signal through the α-receptor of the respective other cytokine. We further create eight chimeric IL-6/IL-11 receptors, which are all biologically active. We find that the domains D1 to D3, which contain the cytokine binding module (CBM), determine which cytokine can activate the chimeric receptor, whereas the stalk region, the transmembrane region, or the intracellular region do not participate in the ligand selectivity of the receptor and are therefore interchangeable between IL-6R and IL-11R. These results suggest a modular organization of the IL-6R and IL-11R, and a similar signal transduction complex of the two cytokines.
Subject(s)
Interleukin-11 Receptor alpha Subunit/chemistry , Interleukin-6 Receptor alpha Subunit/chemistry , Models, Molecular , Receptors, Interleukin-6/chemistry , Animals , Binding Sites , Cell Line , Cell Proliferation , Cytokine Receptor gp130/agonists , Cytokine Receptor gp130/chemistry , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/metabolism , Humans , Interleukin-11/genetics , Interleukin-11/metabolism , Interleukin-11 Receptor alpha Subunit/agonists , Interleukin-11 Receptor alpha Subunit/genetics , Interleukin-11 Receptor alpha Subunit/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-6 Receptor alpha Subunit/agonists , Interleukin-6 Receptor alpha Subunit/genetics , Interleukin-6 Receptor alpha Subunit/metabolism , Ligands , Mice , Peptide Fragments/agonists , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Protein Subunits , Receptors, Interleukin-6/agonists , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Signal TransductionABSTRACT
Interleukin (IL)-6, the founding member of IL-6 family cytokines, plays non-redundant roles in hematopoiesis and acute phase responses. IL-6 signals via a specific private IL-6Rα and a common beta chain gp130. In this study, we have cloned both the IL-6Rα and gp130 in rainbow trout. The trout gp130 cDNA encodes 906 aa and is similar in size, extracellular domain structure (D1-D6) and presence of intracellular motifs important for signal transduction to tetrapod gp130s. The trout IL-6Rα cDNA encodes for 834 aa and is larger compared to tetrapod IL-6Rαs, as are other fish IL-6Rα molecules due to a large D1 domain. However, the cytokine-binding domain is well conserved across vertebrates, with four conserved cysteine residues in the N-terminal FNIII domain and a WSXWS motif in the C-terminal FNIII domain. Furthermore, a phylogenetic tree analysis confirmed that the reported fish IL-6Rα and gp130 molecules are orthologues to their tetrapod counterparts. The extra large D1 domain of the salmonid IL-6Rα molecules results partially from the insertions of two repetitive sequences of [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR], respectively. Furthermore the numbers of repetitions of the two motifs were variable in different individuals and cell lines, and even in the same fish allelic polymorphism exists. Trout IL-6Rα was expressed at higher levels than gp130 in a number of tissues examined and the expression of both IL-6Rα and gp130 could be modulated by LPS and Poly I:C in the cell lines studied. The expression patterns of the receptors suggest that high level expression of IL-6Rα is critical for IL-6 responsiveness.
Subject(s)
Cytokine Receptor gp130/genetics , Interleukin-6 Receptor alpha Subunit/genetics , Oncorhynchus mykiss/genetics , Oncorhynchus mykiss/immunology , Amino Acid Sequence , Animals , Cell Line , Cytokine Receptor gp130/chemistry , Cytokine Receptor gp130/metabolism , Gene Expression Regulation , Interleukin-6 Receptor alpha Subunit/chemistry , Interleukin-6 Receptor alpha Subunit/metabolism , Molecular Sequence Data , Oncorhynchus mykiss/classification , Organ Specificity/genetics , Phylogeny , Polymorphism, Genetic , Protein Structure, Tertiary , Repetitive Sequences, Amino Acid , Sequence Alignment , Sequence Analysis, DNAABSTRACT
To investigate the function of the N-terminal immunoglobulin (Ig)-like domain of the human interleukin-6 receptor alpha-chain (hIL-6R), we constructed a soluble human interleukin-6 receptor (shIL-6R) (named EC05, amino acids 20-354) and soluble variants of the shIL-6R lacking the Ig-like domain (named EC70, amino acids 105-354). The two extracellular portions of hIL-6R were expressed as soluble fusion proteins with thioredoxin in Escherichia coli and purified by using Ni-NTA agarose. Western blot showed that purified proteins were immunoreactive with the antibody against hIL-6R. They also possessed specific binding activity with human interleukin-6 (hIL-6) in ELISA analysis.
