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

Larsen JV, Kristensen AM, Pallesen LT, Bauer J, Vægter CB, Nielsen MS, Madsen P, Petersen CM - Mol. Cell. Biol. (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The MIND Center, Department of Biomedicine, Aarhus University, Aarhus, Denmark jvl@biomed.au.dk cmp@biomed.au.dk.

No MeSH data available.


Related in: MedlinePlus

Interaction between the luminal domains of CNTFRα and sorLA. (A) Concentration-dependent binding of sCNTFRα to the immobilized luminal (entire extracellular) part of sorLA. (B) RAP-mediated inhibition of the interaction between sCNTFRα and sorLA. Immobilized sorLA was exposed to unsupplemented buffer (dotted line) or to buffer supplemented with 5 μM RAP (solid line) prior to the addition of sCNTFRα (arrows). (C) Concentration dependence of sCNTFRα binding to the immobilized Vps10p-D of sorLA. (D) Cross-linking of full-length receptors in cells. HEK293 double transfectants expressing CNTFRα-Myc and sorLA were incubated with the chemical cross-linker DSP (45 min, 2 nM), and receptor proteins were precipitated from lysates using anti-Myc and anti-sorLA antibodies as indicated. Reduced (+DTT) and unreduced samples of precipitate were analyzed by SDS-PAGE. IP, immunoprecipitation; ab, antibody.
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Figure 6: Interaction between the luminal domains of CNTFRα and sorLA. (A) Concentration-dependent binding of sCNTFRα to the immobilized luminal (entire extracellular) part of sorLA. (B) RAP-mediated inhibition of the interaction between sCNTFRα and sorLA. Immobilized sorLA was exposed to unsupplemented buffer (dotted line) or to buffer supplemented with 5 μM RAP (solid line) prior to the addition of sCNTFRα (arrows). (C) Concentration dependence of sCNTFRα binding to the immobilized Vps10p-D of sorLA. (D) Cross-linking of full-length receptors in cells. HEK293 double transfectants expressing CNTFRα-Myc and sorLA were incubated with the chemical cross-linker DSP (45 min, 2 nM), and receptor proteins were precipitated from lysates using anti-Myc and anti-sorLA antibodies as indicated. Reduced (+DTT) and unreduced samples of precipitate were analyzed by SDS-PAGE. IP, immunoprecipitation; ab, antibody.

Mentions: Whereas the former result confirmed that CLC:CLF-1 may bind both receptors simultaneously and thus promote sorLA-mediated endocytosis of CNTFRα, the latter seemed to suggest that the mere presence of sorLA was enough to facilitate a significant degree of endocytosis and that this effect was blocked by CLC, which binds CNTFRα but not sorLA (Fig. 1E). To clarify this, we first performed SPR analysis to determine if the ectodomains of the two receptors interacted. Soluble CNTFRα bound to immobilized sorLA and competed with RAP for binding (Fig. 6A and B). RAP interacts with both the complement-type repeats and the Vps10p-D of sorLA, but as further analysis demonstrated specific binding of sCNTFRα to the purified Vps10p-D (Fig. 6C), this domain must harbor the binding site. Since sCNTFRα in complex with CLC did not associated with sorLA (Fig. 2A), it can be deduced that the two receptors can interact directly in the absence of ligands or interact indirectly (by proxy) in the presence of CLC:CLF-1 and that CLC, when complexed to CNTFRα, inhibits/blocks the interaction.


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

Larsen JV, Kristensen AM, Pallesen LT, Bauer J, Vægter CB, Nielsen MS, Madsen P, Petersen CM - Mol. Cell. Biol. (2016)

Interaction between the luminal domains of CNTFRα and sorLA. (A) Concentration-dependent binding of sCNTFRα to the immobilized luminal (entire extracellular) part of sorLA. (B) RAP-mediated inhibition of the interaction between sCNTFRα and sorLA. Immobilized sorLA was exposed to unsupplemented buffer (dotted line) or to buffer supplemented with 5 μM RAP (solid line) prior to the addition of sCNTFRα (arrows). (C) Concentration dependence of sCNTFRα binding to the immobilized Vps10p-D of sorLA. (D) Cross-linking of full-length receptors in cells. HEK293 double transfectants expressing CNTFRα-Myc and sorLA were incubated with the chemical cross-linker DSP (45 min, 2 nM), and receptor proteins were precipitated from lysates using anti-Myc and anti-sorLA antibodies as indicated. Reduced (+DTT) and unreduced samples of precipitate were analyzed by SDS-PAGE. IP, immunoprecipitation; ab, antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Interaction between the luminal domains of CNTFRα and sorLA. (A) Concentration-dependent binding of sCNTFRα to the immobilized luminal (entire extracellular) part of sorLA. (B) RAP-mediated inhibition of the interaction between sCNTFRα and sorLA. Immobilized sorLA was exposed to unsupplemented buffer (dotted line) or to buffer supplemented with 5 μM RAP (solid line) prior to the addition of sCNTFRα (arrows). (C) Concentration dependence of sCNTFRα binding to the immobilized Vps10p-D of sorLA. (D) Cross-linking of full-length receptors in cells. HEK293 double transfectants expressing CNTFRα-Myc and sorLA were incubated with the chemical cross-linker DSP (45 min, 2 nM), and receptor proteins were precipitated from lysates using anti-Myc and anti-sorLA antibodies as indicated. Reduced (+DTT) and unreduced samples of precipitate were analyzed by SDS-PAGE. IP, immunoprecipitation; ab, antibody.
Mentions: Whereas the former result confirmed that CLC:CLF-1 may bind both receptors simultaneously and thus promote sorLA-mediated endocytosis of CNTFRα, the latter seemed to suggest that the mere presence of sorLA was enough to facilitate a significant degree of endocytosis and that this effect was blocked by CLC, which binds CNTFRα but not sorLA (Fig. 1E). To clarify this, we first performed SPR analysis to determine if the ectodomains of the two receptors interacted. Soluble CNTFRα bound to immobilized sorLA and competed with RAP for binding (Fig. 6A and B). RAP interacts with both the complement-type repeats and the Vps10p-D of sorLA, but as further analysis demonstrated specific binding of sCNTFRα to the purified Vps10p-D (Fig. 6C), this domain must harbor the binding site. Since sCNTFRα in complex with CLC did not associated with sorLA (Fig. 2A), it can be deduced that the two receptors can interact directly in the absence of ligands or interact indirectly (by proxy) in the presence of CLC:CLF-1 and that CLC, when complexed to CNTFRα, inhibits/blocks the interaction.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The MIND Center, Department of Biomedicine, Aarhus University, Aarhus, Denmark jvl@biomed.au.dk cmp@biomed.au.dk.

No MeSH data available.


Related in: MedlinePlus