Cytokine-Like Factor 1, an Essential Facilitator of Cardiotrophin-Like Cytokine:Ciliary Neurotrophic Factor Receptor α Signaling and sorLA-Mediated Turnover

Abstract

Cardiotrophin-like cytokine:cytokine-like factor-1 (CLC:CLF-1) is a heterodimeric neurotropic cytokine that plays a crucial role during neuronal development. Mice lacking CLC:CLF-1 die soon after birth due to a suckling defect and show reduced numbers of motor neurons. Humans carrying mutations in CLC:CLF-1 develop similar disorders, known as Sohar-Crisponi or cold-induced sweating syndrome, and have a high risk of early death. It is well known that CLC binds the ciliary neurotrophic factor receptor α (CNTFRα) and is a prerequisite for signaling through the gp130/leukemia inhibitory factor receptor β (LIFRβ) heterodimer, whereas CLF-1 serves to promote the cellular release of CLC. However, the precise role of CLF-1 is unclear. Here, we report that CLF-1, based on its binding site for CLC and on two additional and independent sites for CNTFRα and sorLA, is a key player in CLC and CNTFRα signaling and turnover. The site for CNTFRα enables CLF-1 to promote CLC:CNTFRα complex formation and signaling. The second site establishes a link between the endocytic receptor sorLA and the tripartite CLC:CLF-1:CNTFRα complex and allows sorLA to downregulate the CNTFRα pool in stimulated cells. Finally, sorLA may bind and concentrate the tripartite soluble CLC:CLF-1:CNTFRα complex on cell membranes and thus facilitate its signaling through gp130/LIFRβ.

Crisponi/cold‐induced sweating syndrome: Differential diagnosis, pathogenesis and treatment concepts

“…Furthermore, defective conversion from adrenergic to cholinergic phenotype might explain the bone deformities and muscle symptoms, and the high variability in the clinical presentation of CS/CISS in different stages of life, because cholinergic sympathetic neurons also innervate periosteum and vasculature of the skeletal muscles. However, precise function of CRLF1 in the tripartite complex with CNTFR and CLCF1 is poorly investigated, and recent findings even indicate that CRLF1 might have distinct functions and different interaction partners such as IL 28, SorLA or IL12 . Thus, CRLF1 might have other functions and may target so far unidentified receptors.…”

SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

“…10. Subject an equal amount of protein from each sample to SDS-PAGE 13 and Western blotting 13 and visualize the content of pSTAT3 and total STAT3 in the cell lysates using rabbit anti-pSTAT3 (1:2000) and mouse anti-STAT3 (1:1,000) antibodies and the appropriate HRP-linked mouse and rabbit secondary antibodies (1:2,000). 11.…”

“…Thus, it appears that CLF-1 contains three independent binding sites: one for its well-known binding to CLC; one that mediates direct binding to the CNTFRα; and a third (high affinity) site for interaction with the endocytic receptor sorLA. As both CLC and CLF-1 seem to target CNTFRα with a considerable lower affinity than the CLC:CLF-1 complex, it is conceivable that CLF-1 (via its CNTFRα-binding site) promotes the unification of CLC and CNTFRα and thereby facilitates signaling 13 .…”

“…Its cytoplasmic tail interacts with several adaptor proteins, e.g., adaptor protein-1 and -2, and sorLA conveys efficient endocytosis as well as intracellular sorting and transport of bound proteins [16][17][18] . The Vps10p-domain comprises a large ten-bladed β-propeller 19,20 , which binds a series of unrelated ligands including CLF-1 (but notably, not CLC) 13 . The binding site in CLF-1 is accessible even after complex formation with CLC and CNTFRα which means that sorLA binds not only free CLF-1, but also CLC:CLF-1 and the trimeric complex CLC:CLF-1:CNTFRα CLF-1 and CLC:CLF-1, but these are soluble proteins whereas CNTFRα is fixed to the surface membrane by a GPI-anchor.…”

“…9. Subject an equal amount of protein from each sample to reducing SDS-PAGE 13 and Western blotting 13 and visualize the content of sorLA, β-actin, and CNTFRα-Myc in the medium and in the cell lysates using rabbit anti-sorLA 16 (5 μg/mL), mouse anti-β-actin (0.4 μg/mL), and mouse anti-Myc (1 μg/mL) antibodies and the appropriate HRP-linked secondary antibodies (1:2,000). 10.…”