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

Abstract

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.

Craniosynostosis‐associated variants in the IL‐11R complex: new insights and questions

“…More recent work showed that this variant of IL-11R transfected into HEK293 cells exhibited retention of IL-11R in the endoplasmic reticulum (ER) due to failure of transport to the cell membrane, and that this may have originated in defective post-translational glycosylation of the receptor [6]. Defective glycosylation and trafficking have been studied in a similar manner and proposed for the recently described IL-11R T306_S308dup variant [7]. Such post-translational modifications occur in the ER and are common to many transmembrane proteins.…”

“…Confirming that these most likely relate to loss of function, null mice for IL-11R also exhibit a partially penetrant craniosynostosis phenotype [3,6]. Recent work from Birte Kespohl et al [7] has provided new insights into how some of these variants influence IL-11R function.…”

“…The multiple variants in both IL11RA and IL6ST associated with craniosynostosis point to the complexity of processes required for formation of the IL-11R signalling complex. To date, three potential mechanisms that impair IL-11R signalling have been associated with craniosynostosis: disruption of IL-11R near the IL-11 ligand binding site [3], disruption of gp130 function leading to cytokine-specific signalling defects [13][14][15], and disruption of IL-11R post-translational modifications required for receptor migration to the cell membrane [6,7]. Given the complexity of the IL-11R signalling complex and the mechanisms required for all components to interact appropriately at the cell surface, there may be more.…”

“…Domain 3 also contains a highly conserved tryptophan-arginine ladder; this is a hotspot for craniosynostosis variants (Fig. 1), including W307R [17] and T306_S308dup [7] which both exhibit defects in receptor transport to the cell membrane. The tryptophan-arginine ladder also contains the WSXWS motif which is common to many type-I cytokine receptors and has been proposed to function as a stabiliser for complex formation [18].…”

“…Clearly, this is different for IL-11R. Post-translational modifications required to support receptor folding and transport may also include C-mannosylation, since Kespohl et al [7] show that IL-11R is natively Cmannosylated.…”