Molecular characterization of the craniosynostosis-associated interleukin-11 receptor variants p.T306_S308dup and p.E364_V368del.

Interleukin-11 (IL-11) is a member of the IL-6 family of cytokines and is an important factor for bone homeostasis. IL-11 binds to and signals via the membrane-bound IL-11 receptor (IL-11R, classic signaling) or soluble forms of the IL-11R (sIL-11R, trans-signaling). Mutations in the IL11RA gene, which encodes the IL-11R, are associated with craniosynostosis, a human condition in which one or several of the sutures close prematurely, resulting in malformation of the skull. The biological mechanisms of how mutations within the IL-11R are linked to craniosynostosis are mostly unexplored. In this study, we analyze two variants of the IL-11R described in craniosynostosis patients: p.T306_S308dup, which results in a duplication of three amino-acid residues within the membrane-proximal fibronectin type III domain, and p.E364_V368del, which results in a deletion of five amino-acid residues in the so-called stalk region adjacent to the plasma membrane. The stalk region connects the three extracellular domains to the transmembrane and intracellular region of the IL-11R and contains cleavage sites for different proteases that generate sIL-11R variants. Using a combination of bioinformatics and different biochemical, molecular, and cell biology methods, we show that the IL-11R-T306_S308dup variant does not mature correctly, is intracellularly retained, and does not reach the cell surface. In contrast, the IL-11R-E364_V368del variant is fully biologically active and processed normally by proteases, thus allowing classic and trans-signaling of IL-11. Our results provide evidence that mutations within the IL11RA gene may not be causative for craniosynostosis and suggest that other regulatory mechanism(s) are involved but remain to be identified.

Interleukin-6-interleukin-11 receptor chimeras reveal ionomycin-induced proteolysis beyond ADAM10.

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are two important pleiotropic cytokines, both of which signal through a homodimer of the ß-receptor gp130. Specificity is gained through the unique, nonsignaling α-receptors IL-6R and IL-11R. Soluble variants of IL-6R and IL-11R also exist. Both membrane-bound receptors can be cleaved by the metalloprotease ADAM10. Here, we use ten different chimeric receptors consisting of different parts of IL-6R and IL-11R and analyze their susceptibility toward cleavage by ADAM10. As expected, all chimeras are substrates of ADAM10. However, we observed that cleavage of chimeric receptors containing the stalk region of the IL-11R could be blocked by the protease inhibitor GI (selective for ADAM10), but not by the protease inhibitor GW (selective for both ADAM10 and ADAM17), suggesting that another protease besides ADAM10 is involved in cleavage of these chimeras.

Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling.

Interleukin-11 (IL-11) is a pleiotropic cytokine with both pro- and anti-inflammatory properties. It activates its target cells via binding to the membrane-bound IL-11 receptor (IL-11R), which then recruits a homodimer of the ubiquitously expressed, signal-transducing receptor gp130. Besides this classic signaling pathway, IL-11 can also bind to soluble forms of the IL-11R (sIL-11R), and IL-11/sIL-11R complexes activate cells via the induction of gp130 homodimerization (trans-signaling). We have previously reported that the metalloprotease ADAM10 cleaves the membrane-bound IL-11R and thereby generates sIL-11R. In this study, we identify the rhomboid intramembrane protease RHBDL2 as a so far unrecognized alternative sheddase that can efficiently trigger IL-11R secretion. We determine the cleavage site used by RHBDL2, which is located in the extracellular part of the receptor in close proximity to the plasma membrane, between Ala-370 and Ser-371. Furthermore, we identify critical amino acid residues within the transmembrane helix that are required for IL-11R proteolysis. We also show that ectopically expressed RHBDL2 is able to cleave the IL-11R within the early secretory pathway and not only at the plasma membrane, indicating that its subcellular localization plays a central role in controlling its activity. Moreover, RHBDL2-derived sIL-11R is biologically active and able to perform IL-11 trans-signaling. Finally, we show that the human mutation IL-11R-A370V does not impede IL-11 classic signaling, but prevents RHBDL2-mediated IL-11R cleavage.

Differences in Shedding of the Interleukin-11 Receptor by the Proteases ADAM9, ADAM10, ADAM17, Meprin α, Meprin ß and MT1-MMP.

Interleukin-11 (IL-11) has been associated with inflammatory conditions, bone homeostasis, hematopoiesis, and fertility. So far, these functions have been linked to classical IL-11 signaling via the membrane bound receptor (IL-11R). However, a signaling cascade via the soluble IL-11R (sIL-11R), generated by proteolytic cleavage, can also be induced. This process is called IL-11 trans-signaling. A disintegrin and metalloprotease 10 (ADAM10) and neutrophil elastase were described as ectodomain sheddases of the IL-11R, thereby inducing trans-signaling. Furthermore, previous studies employing approaches for the stimulation and inhibition of endogenous ADAM-proteases indicated that ADAM10, but not ADAM17, can cleave the IL-11R. Herein, we show that several metalloproteases, namely ADAM9, ADAM10, ADAM17, meprin ß, and membrane-type 1 matrix metalloprotease/matrix metalloprotease-14 (MT1-MMP/MMP-14) when overexpressed are able to shed the IL-11R. All sIL-11R ectodomains were biologically active and capable of inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation in target cells. The difference observed for ADAM10/17 specificity compared to previous studies can be explained by the different approaches used, such as stimulation of protease activity or making use of cells with genetically deleted enzymes.

Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor.

BACKGROUND/AIMS: The cytokine interleukin-11 (IL-11) has important pro- and anti-inflammatory functions. It activates its target cells through binding to the IL-11 receptor (IL-11R), and the IL-11/IL-11R complex recruits a homodimer of glycoprotein 130 (gp130). N-linked glycosylation, a post-translational modification where complex oligosaccharides are attached to the side chain of asparagine residues, is often important for stability, folding and biological function of cytokine receptors. METHODS: We generated different IL-11R mutants via site-directed mutagenesis and analyzed them in different cell lines via Western blot, flow cytometry, confocal microscopy and proliferation assays. RESULTS: In this study, we identified two functional N-glycosylation sites in the D2 domain of the IL-11R at N127 and N194. While mutation of N127Q only slightly affects cell surface expression of the IL-11R, mutation of N194Q broadly prevents IL-11R appearance at the plasma membrane. Accordingly, IL-11R mutants lacking N194 are retained within the ER, whereas the N127 mutant is transported through the Golgi complex to the cell surface, uncovering a differential role of the two N-glycan sequons for IL-11R maturation. Interestingly, IL-11R mutants devoid of one or both N-glycans are still biologically active. Furthermore, the IL-11RN127Q/N194Q mutant shows no inducible shedding by ADAM10, but is rather constitutively released into the supernatant. CONCLUSION: Our results show that the two N-glycosylation sites differentially influence stability and proteolytic processing of the IL-11R, but that N-linked glycosylation is not a prerequisite for IL-11 signaling.

The SNP rs4252548 (R112H) which is associated with reduced human height compromises the stability of IL-11.

Height is a complex human phenotype that is influenced by variations in a high number of genes. Recently, a single nucleotide polymorphism (SNP) within IL11 (rs4252548) has been described to be associated with height in adults of European ancestry. This coding SNP leads to the exchange of Arg-112 to His-112 within the cytokine Interleukin-11 (IL-11), which has a well-established role in osteoclast development and bone turnover. The functional consequences of the R112H mutation are unknown so far. In this study, we show by molecular replacement that Arg-112 does not participate in binding of IL-11 to its receptors IL-11R and glycoprotein 130 (gp130). Recombinant IL-11 R112H expressed in E. coli displays a correct four-helix-bundle folding topology, and binds with similar affinity to IL-11R and the IL-11/IL-11R/gp130 complex. IL-11 R112H induces cell proliferation and phosphorylation of the downstream transcription factor STAT3 indistinguishable from IL-11. However, IL-11 R112H fails to support the survival of osteoclast progenitor cells and is less thermally stable, which is caused by the loss of the positive charge on the protein surface since protonation of the histidine side chain recovers stability.

The dynamics of signal triggering in a gp130-receptor complex.

gp130 is a shared signal-transducing membrane-associated receptor for several hematopoietic cytokines. The 30 A resolution cryo-electron microscopy (cryo-EM) structure of the Interleukin 11(IL-11)-IL-11 Receptor-gp130 extracellular complex reveals the architecture and dynamics of this gp130-containing signaling complex. Normal-mode analysis reveals a repertoire of conformational changes that could function in signal triggering. This suggests a concerted mechanism of signaling involving all the components of the complex. This could provide a general mechanism of signal transfer for cytokines utilizing the JAK-STAT signaling cascade.