Limits...
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

sorLA-mediated internalization of CNTFRα. (A) HEK293 cells transfected with Myc-tagged CNTFRα or doubly transfected with CNTFRα-Myc and sorLA were incubated (37°C, 10 min) with mouse anti-Myc, washed, and then incubated with the given ligands (at 10 nM). At the given times the cells were fixed, permeabilized, and stained with Alexa Fluor 488-conjugated goat anti-mouse Ig. (B) Internalization and colocalization of CNTFRα (endogenous) and sorLA in SH-SY5Y transfectants. Wild-type cells and sorLA transfectants were incubated (37°C for 25 min) with the given ligands (at 10 nM) and then fixed, washed, permeabilized, and finally stained by goat anti-CNTFRα and mouse anti-sorLA. Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated donkey anti-mouse antibodies were used as secondary antibodies. (C) Number of CNTFRα-containing vesicles in wt and sorLA transfected SH-SY5Y cells. The cells were incubated with ligands and stained as described for panel B prior to automated counting of vesicles with a positive stain for CNTFRα. Each column represents results obtained in 346 to 2,047 cells (containing between 800 and 9,444 positive vesicles) and shows mean values ± SEMs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836274&req=5

Figure 5: sorLA-mediated internalization of CNTFRα. (A) HEK293 cells transfected with Myc-tagged CNTFRα or doubly transfected with CNTFRα-Myc and sorLA were incubated (37°C, 10 min) with mouse anti-Myc, washed, and then incubated with the given ligands (at 10 nM). At the given times the cells were fixed, permeabilized, and stained with Alexa Fluor 488-conjugated goat anti-mouse Ig. (B) Internalization and colocalization of CNTFRα (endogenous) and sorLA in SH-SY5Y transfectants. Wild-type cells and sorLA transfectants were incubated (37°C for 25 min) with the given ligands (at 10 nM) and then fixed, washed, permeabilized, and finally stained by goat anti-CNTFRα and mouse anti-sorLA. Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated donkey anti-mouse antibodies were used as secondary antibodies. (C) Number of CNTFRα-containing vesicles in wt and sorLA transfected SH-SY5Y cells. The cells were incubated with ligands and stained as described for panel B prior to automated counting of vesicles with a positive stain for CNTFRα. Each column represents results obtained in 346 to 2,047 cells (containing between 800 and 9,444 positive vesicles) and shows mean values ± SEMs.

Mentions: The SPR data (Fig. 2B) and the findings in SH-SY5Y cells suggested that the CLC:CLF-1 heterodimer might serve as a molecular link between sorLA and CNTFRα, and this prompted us to examine if sorLA, via binding to CLC:CLF-1, also conveyed uptake of CNTFRα. HEK293 cells expressing CNTFRα-Myc alone or both receptors in combination were fixed and stained with an anti-Myc antibody before and after incubation (37°C) in unsupplemented medium or in medium supplemented with 10 nM CLC or CLC:CLF-1. It appears from the subsequent fluorescence microscopy (Fig. 5A) that in the absence of sorLA nearly all CNTF receptors were localized to the surface membrane both before and after incubation with ligands. In contrast, distinct intracellular staining, signifying internalized CNTFRα, was seen in the double transfectants. Staining was particularly strong in the cells treated with CLC:CLF-1, but minor staining was also seen upon stimulation with CLC and, surprisingly, in cells not subjected to either of the two ligands.


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)

sorLA-mediated internalization of CNTFRα. (A) HEK293 cells transfected with Myc-tagged CNTFRα or doubly transfected with CNTFRα-Myc and sorLA were incubated (37°C, 10 min) with mouse anti-Myc, washed, and then incubated with the given ligands (at 10 nM). At the given times the cells were fixed, permeabilized, and stained with Alexa Fluor 488-conjugated goat anti-mouse Ig. (B) Internalization and colocalization of CNTFRα (endogenous) and sorLA in SH-SY5Y transfectants. Wild-type cells and sorLA transfectants were incubated (37°C for 25 min) with the given ligands (at 10 nM) and then fixed, washed, permeabilized, and finally stained by goat anti-CNTFRα and mouse anti-sorLA. Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated donkey anti-mouse antibodies were used as secondary antibodies. (C) Number of CNTFRα-containing vesicles in wt and sorLA transfected SH-SY5Y cells. The cells were incubated with ligands and stained as described for panel B prior to automated counting of vesicles with a positive stain for CNTFRα. Each column represents results obtained in 346 to 2,047 cells (containing between 800 and 9,444 positive vesicles) and shows mean values ± SEMs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836274&req=5

Figure 5: sorLA-mediated internalization of CNTFRα. (A) HEK293 cells transfected with Myc-tagged CNTFRα or doubly transfected with CNTFRα-Myc and sorLA were incubated (37°C, 10 min) with mouse anti-Myc, washed, and then incubated with the given ligands (at 10 nM). At the given times the cells were fixed, permeabilized, and stained with Alexa Fluor 488-conjugated goat anti-mouse Ig. (B) Internalization and colocalization of CNTFRα (endogenous) and sorLA in SH-SY5Y transfectants. Wild-type cells and sorLA transfectants were incubated (37°C for 25 min) with the given ligands (at 10 nM) and then fixed, washed, permeabilized, and finally stained by goat anti-CNTFRα and mouse anti-sorLA. Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated donkey anti-mouse antibodies were used as secondary antibodies. (C) Number of CNTFRα-containing vesicles in wt and sorLA transfected SH-SY5Y cells. The cells were incubated with ligands and stained as described for panel B prior to automated counting of vesicles with a positive stain for CNTFRα. Each column represents results obtained in 346 to 2,047 cells (containing between 800 and 9,444 positive vesicles) and shows mean values ± SEMs.
Mentions: The SPR data (Fig. 2B) and the findings in SH-SY5Y cells suggested that the CLC:CLF-1 heterodimer might serve as a molecular link between sorLA and CNTFRα, and this prompted us to examine if sorLA, via binding to CLC:CLF-1, also conveyed uptake of CNTFRα. HEK293 cells expressing CNTFRα-Myc alone or both receptors in combination were fixed and stained with an anti-Myc antibody before and after incubation (37°C) in unsupplemented medium or in medium supplemented with 10 nM CLC or CLC:CLF-1. It appears from the subsequent fluorescence microscopy (Fig. 5A) that in the absence of sorLA nearly all CNTF receptors were localized to the surface membrane both before and after incubation with ligands. In contrast, distinct intracellular staining, signifying internalized CNTFRα, was seen in the double transfectants. Staining was particularly strong in the cells treated with CLC:CLF-1, but minor staining was also seen upon stimulation with CLC and, surprisingly, in cells not subjected to either of the two ligands.

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