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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

Cellular uptake of CLC, CLF-1, and CLC:CLF-1. (A) Untransfected and sorLA transfected HEK293 cells were incubated (37°C, 25 min) at 10 nM concentrations of the indicated ligands and then washed, fixed, and permeabilized. The cells were finally stained using mouse anti-His or rabbit anti-CLC as primary antibodies and Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit antibodies as secondary antibodies. (B) Colocalization of internalized CLC:CLF-1 and sorLA. sorLA transfected HEK293 cells were incubated with CLC:CLF-1 as described for panel A and subsequently stained with rabbit anti-CLC, mouse anti-sorLA, and matching secondary antibodies. (C) Colocalization of CLC:CLF-1 and endogenous CNTFRα (10 nM, 25 min). Following fixation the cells were stained with mouse anti-His, goat anti-CNTFRα, and appropriate secondary antibodies (Alexa Fluor 488-conjugated anti-mouse Ig and Alexa Fluor 568-conjugated anti-goat Ig).
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Figure 3: Cellular uptake of CLC, CLF-1, and CLC:CLF-1. (A) Untransfected and sorLA transfected HEK293 cells were incubated (37°C, 25 min) at 10 nM concentrations of the indicated ligands and then washed, fixed, and permeabilized. The cells were finally stained using mouse anti-His or rabbit anti-CLC as primary antibodies and Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit antibodies as secondary antibodies. (B) Colocalization of internalized CLC:CLF-1 and sorLA. sorLA transfected HEK293 cells were incubated with CLC:CLF-1 as described for panel A and subsequently stained with rabbit anti-CLC, mouse anti-sorLA, and matching secondary antibodies. (C) Colocalization of CLC:CLF-1 and endogenous CNTFRα (10 nM, 25 min). Following fixation the cells were stained with mouse anti-His, goat anti-CNTFRα, and appropriate secondary antibodies (Alexa Fluor 488-conjugated anti-mouse Ig and Alexa Fluor 568-conjugated anti-goat Ig).

Mentions: Binding to full-length sorLA was studied in cells. Wild-type HEK293 cells and cells stably transfected with sorLA were incubated at 37°C in medium containing a 10 nM concentration of either CLC, His-tagged CLF-1, or CLC:CLF-1. After 25 min the cells were fixed, permeabilized, and stained with anti-CLC or anti-His tag antibodies to visualize internalized ligands. No staining was observed in untransfected cells, but in good agreement with the SPR results, CLF-1 and CLC:CLF-1, but not CLC, had been taken up (and internalized) by the sorLA transfectants (Fig. 3A). Double staining (for CLC:CLF-1 and sorLA) of cells from similar experiments demonstrated that the internalized ligand colocalized with sorLA (Fig. 3B). Similar results were obtained in sorLA transfected SH-SY5Y cells (data not shown), but in these cells, which express endogenous CNTFRα, internalized CLC:CLF-1 was furthermore seen to colocalize with CNTFRα (Fig. 3C).


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)

Cellular uptake of CLC, CLF-1, and CLC:CLF-1. (A) Untransfected and sorLA transfected HEK293 cells were incubated (37°C, 25 min) at 10 nM concentrations of the indicated ligands and then washed, fixed, and permeabilized. The cells were finally stained using mouse anti-His or rabbit anti-CLC as primary antibodies and Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit antibodies as secondary antibodies. (B) Colocalization of internalized CLC:CLF-1 and sorLA. sorLA transfected HEK293 cells were incubated with CLC:CLF-1 as described for panel A and subsequently stained with rabbit anti-CLC, mouse anti-sorLA, and matching secondary antibodies. (C) Colocalization of CLC:CLF-1 and endogenous CNTFRα (10 nM, 25 min). Following fixation the cells were stained with mouse anti-His, goat anti-CNTFRα, and appropriate secondary antibodies (Alexa Fluor 488-conjugated anti-mouse Ig and Alexa Fluor 568-conjugated anti-goat Ig).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Cellular uptake of CLC, CLF-1, and CLC:CLF-1. (A) Untransfected and sorLA transfected HEK293 cells were incubated (37°C, 25 min) at 10 nM concentrations of the indicated ligands and then washed, fixed, and permeabilized. The cells were finally stained using mouse anti-His or rabbit anti-CLC as primary antibodies and Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit antibodies as secondary antibodies. (B) Colocalization of internalized CLC:CLF-1 and sorLA. sorLA transfected HEK293 cells were incubated with CLC:CLF-1 as described for panel A and subsequently stained with rabbit anti-CLC, mouse anti-sorLA, and matching secondary antibodies. (C) Colocalization of CLC:CLF-1 and endogenous CNTFRα (10 nM, 25 min). Following fixation the cells were stained with mouse anti-His, goat anti-CNTFRα, and appropriate secondary antibodies (Alexa Fluor 488-conjugated anti-mouse Ig and Alexa Fluor 568-conjugated anti-goat Ig).
Mentions: Binding to full-length sorLA was studied in cells. Wild-type HEK293 cells and cells stably transfected with sorLA were incubated at 37°C in medium containing a 10 nM concentration of either CLC, His-tagged CLF-1, or CLC:CLF-1. After 25 min the cells were fixed, permeabilized, and stained with anti-CLC or anti-His tag antibodies to visualize internalized ligands. No staining was observed in untransfected cells, but in good agreement with the SPR results, CLF-1 and CLC:CLF-1, but not CLC, had been taken up (and internalized) by the sorLA transfectants (Fig. 3A). Double staining (for CLC:CLF-1 and sorLA) of cells from similar experiments demonstrated that the internalized ligand colocalized with sorLA (Fig. 3B). Similar results were obtained in sorLA transfected SH-SY5Y cells (data not shown), but in these cells, which express endogenous CNTFRα, internalized CLC:CLF-1 was furthermore seen to colocalize with CNTFRα (Fig. 3C).

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