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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

sorLA mediates CLC:CLF-1-dependent downregulation of CNTFRα. (A and B) HEK293 single transfectants expressing CNTFRα-Myc (A) and double transfectants expressing both CNTFRα-Myc and sorLA (B) were incubated in the absence (−) or presence (+) of 10 nM CLC:CLF-1. Incubation was stopped (time zero or after 5 h), and the content of CNTFRα-Myc found in the medium (m) and in cell lysates (l) was detected by Western blotting and quantified by densitometry of specific bands. Upper panels show Western blot results from a typical experiment. Lower panels show the detected levels of CNTFRα-Myc found in cell lysates. The levels are shown relative to the CNTFRα-Myc level in single transfectants at time zero. Data represent means ± SEMs of three experiments. (C) Western blotting and quantitation of CNTFRα in cells subjected or not to lysosomal inhibitors prior to incubation (5 h) with CLC:CLF-1. (D) Immunofluorescence showing accumulation of CNTFRα in LAMP-1-positive vesicles of cells treated with lysosomal inhibitors. (E) HEK293 cells were preincubated in the absence or presence of 10 nM CLC:CLF-1 for 5 h (pre-exp.), washed, starved in unsupplemented medium for 1.5 h, and finally restimulated (stim.) with 5 nM CLC:CLF-1, as indicated, for 15 min. The columns show the relative levels of pSTAT3 in wt HEK293 cells and in sorLA transfectants. Data represent means ± SEMs (n = 3) relative to the pSTAT3 level in cells preincubated in the absence of CLC:CLF-1 but restimulated with CLC:CLF-1. The right panel shows a Western blot of the response (pSTAT3) obtained in an experiment with cells overexpressing sorLA.
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Figure 7: sorLA mediates CLC:CLF-1-dependent downregulation of CNTFRα. (A and B) HEK293 single transfectants expressing CNTFRα-Myc (A) and double transfectants expressing both CNTFRα-Myc and sorLA (B) were incubated in the absence (−) or presence (+) of 10 nM CLC:CLF-1. Incubation was stopped (time zero or after 5 h), and the content of CNTFRα-Myc found in the medium (m) and in cell lysates (l) was detected by Western blotting and quantified by densitometry of specific bands. Upper panels show Western blot results from a typical experiment. Lower panels show the detected levels of CNTFRα-Myc found in cell lysates. The levels are shown relative to the CNTFRα-Myc level in single transfectants at time zero. Data represent means ± SEMs of three experiments. (C) Western blotting and quantitation of CNTFRα in cells subjected or not to lysosomal inhibitors prior to incubation (5 h) with CLC:CLF-1. (D) Immunofluorescence showing accumulation of CNTFRα in LAMP-1-positive vesicles of cells treated with lysosomal inhibitors. (E) HEK293 cells were preincubated in the absence or presence of 10 nM CLC:CLF-1 for 5 h (pre-exp.), washed, starved in unsupplemented medium for 1.5 h, and finally restimulated (stim.) with 5 nM CLC:CLF-1, as indicated, for 15 min. The columns show the relative levels of pSTAT3 in wt HEK293 cells and in sorLA transfectants. Data represent means ± SEMs (n = 3) relative to the pSTAT3 level in cells preincubated in the absence of CLC:CLF-1 but restimulated with CLC:CLF-1. The right panel shows a Western blot of the response (pSTAT3) obtained in an experiment with cells overexpressing sorLA.

Mentions: To determine if sorLA-mediated internalization of the CLC:CLF-1:CNTFRα complex was accompanied by downregulation (degradation) of CNTFRα, we initially tried to detect downregulation using biolabeled receptors. However, as labeling was poor due to a very modest synthesis of CNTFRα at steady state, we instead decided to measure the total pool of CNTFRα in HEK293 transfectants before and after incubation with CLC:CLF-1. Single transfectants expressing CNTFRα-Myc and double transfectants expressing both CNTFRα-Myc and sorLA were incubated at 10 nM CLC:CLF-1 or in unsupplemented medium. After 0 and 5 h of incubation, the cells were lysed, and their content of CNTFRα was quantified by Western blotting. The results are shown in Fig. 7, and it appears (Fig. 7B) that in the double transfectants the cellular pool of CNTFRα was reduced by more than 50% upon 5 h of exposure to CLC:CLF-1. In contrast, the bulk of CNTFRα in the single transfectants (subjected to CLC:CLF-1 or not) remained virtually unchanged (showing a slight but insignificant increase) upon incubation with CLC:CLF-1 (Fig. 7A). Furthermore, downregulation of CNTFRα was almost absent in cells treated with the lysosomal inhibitors leupeptin and pepstatin A (Fig. 7C) and was instead seen to accumulate in LAMP-1-positive vesicles (Fig. 7D). Thus, our data demonstrate that CNTFRα, following endocytosis in complex with CLC:CLF-1 and sorLA, is directed toward degradation in lysosomes rather than recycled to the surface membrane.


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 mediates CLC:CLF-1-dependent downregulation of CNTFRα. (A and B) HEK293 single transfectants expressing CNTFRα-Myc (A) and double transfectants expressing both CNTFRα-Myc and sorLA (B) were incubated in the absence (−) or presence (+) of 10 nM CLC:CLF-1. Incubation was stopped (time zero or after 5 h), and the content of CNTFRα-Myc found in the medium (m) and in cell lysates (l) was detected by Western blotting and quantified by densitometry of specific bands. Upper panels show Western blot results from a typical experiment. Lower panels show the detected levels of CNTFRα-Myc found in cell lysates. The levels are shown relative to the CNTFRα-Myc level in single transfectants at time zero. Data represent means ± SEMs of three experiments. (C) Western blotting and quantitation of CNTFRα in cells subjected or not to lysosomal inhibitors prior to incubation (5 h) with CLC:CLF-1. (D) Immunofluorescence showing accumulation of CNTFRα in LAMP-1-positive vesicles of cells treated with lysosomal inhibitors. (E) HEK293 cells were preincubated in the absence or presence of 10 nM CLC:CLF-1 for 5 h (pre-exp.), washed, starved in unsupplemented medium for 1.5 h, and finally restimulated (stim.) with 5 nM CLC:CLF-1, as indicated, for 15 min. The columns show the relative levels of pSTAT3 in wt HEK293 cells and in sorLA transfectants. Data represent means ± SEMs (n = 3) relative to the pSTAT3 level in cells preincubated in the absence of CLC:CLF-1 but restimulated with CLC:CLF-1. The right panel shows a Western blot of the response (pSTAT3) obtained in an experiment with cells overexpressing sorLA.
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Figure 7: sorLA mediates CLC:CLF-1-dependent downregulation of CNTFRα. (A and B) HEK293 single transfectants expressing CNTFRα-Myc (A) and double transfectants expressing both CNTFRα-Myc and sorLA (B) were incubated in the absence (−) or presence (+) of 10 nM CLC:CLF-1. Incubation was stopped (time zero or after 5 h), and the content of CNTFRα-Myc found in the medium (m) and in cell lysates (l) was detected by Western blotting and quantified by densitometry of specific bands. Upper panels show Western blot results from a typical experiment. Lower panels show the detected levels of CNTFRα-Myc found in cell lysates. The levels are shown relative to the CNTFRα-Myc level in single transfectants at time zero. Data represent means ± SEMs of three experiments. (C) Western blotting and quantitation of CNTFRα in cells subjected or not to lysosomal inhibitors prior to incubation (5 h) with CLC:CLF-1. (D) Immunofluorescence showing accumulation of CNTFRα in LAMP-1-positive vesicles of cells treated with lysosomal inhibitors. (E) HEK293 cells were preincubated in the absence or presence of 10 nM CLC:CLF-1 for 5 h (pre-exp.), washed, starved in unsupplemented medium for 1.5 h, and finally restimulated (stim.) with 5 nM CLC:CLF-1, as indicated, for 15 min. The columns show the relative levels of pSTAT3 in wt HEK293 cells and in sorLA transfectants. Data represent means ± SEMs (n = 3) relative to the pSTAT3 level in cells preincubated in the absence of CLC:CLF-1 but restimulated with CLC:CLF-1. The right panel shows a Western blot of the response (pSTAT3) obtained in an experiment with cells overexpressing sorLA.
Mentions: To determine if sorLA-mediated internalization of the CLC:CLF-1:CNTFRα complex was accompanied by downregulation (degradation) of CNTFRα, we initially tried to detect downregulation using biolabeled receptors. However, as labeling was poor due to a very modest synthesis of CNTFRα at steady state, we instead decided to measure the total pool of CNTFRα in HEK293 transfectants before and after incubation with CLC:CLF-1. Single transfectants expressing CNTFRα-Myc and double transfectants expressing both CNTFRα-Myc and sorLA were incubated at 10 nM CLC:CLF-1 or in unsupplemented medium. After 0 and 5 h of incubation, the cells were lysed, and their content of CNTFRα was quantified by Western blotting. The results are shown in Fig. 7, and it appears (Fig. 7B) that in the double transfectants the cellular pool of CNTFRα was reduced by more than 50% upon 5 h of exposure to CLC:CLF-1. In contrast, the bulk of CNTFRα in the single transfectants (subjected to CLC:CLF-1 or not) remained virtually unchanged (showing a slight but insignificant increase) upon incubation with CLC:CLF-1 (Fig. 7A). Furthermore, downregulation of CNTFRα was almost absent in cells treated with the lysosomal inhibitors leupeptin and pepstatin A (Fig. 7C) and was instead seen to accumulate in LAMP-1-positive vesicles (Fig. 7D). Thus, our data demonstrate that CNTFRα, following endocytosis in complex with CLC:CLF-1 and sorLA, is directed toward degradation in lysosomes rather than recycled to the surface membrane.

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