Claus Munck Petersen

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

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SorLA and CLC : CLF-1-dependent Downregulation of CNTFR alpha as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry. / Larsen, Jakob V.; Petersen, Claus M.

In: Journal of Visualized Experiments, No. 119, 01.2017.

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@article{3a0aceb4b4894829918203997a3f5fc6,
title = "SorLA and CLC: CLF-1-dependent Downregulation of CNTFR alpha as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry",
abstract = "The heterodimeric cytokine Cardiotrophin-like Cytokine: Cytokine-like Factor-1 (CLC: CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFR alpha to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-beta (gp130/LIFR beta) for signaling. Both CLC and CNTFR alpha are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFR alpha (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC: CLF-1, as well as the trimeric (CLC: CLF-1: CNTFR alpha) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC: CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFR alpha and sorLA, CNTFR alpha first binds CLC: CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFR alpha, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC: CLF-1. The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC: CLF-1-dependent downregulation of surface-membrane CNTFR alpha expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFR alpha; and iii) the lowered cellular response to CLC: CLF-1-stimulation upon sorLA-mediated downregulation of CNTFR alpha.",
keywords = "Biochemistry, Issue 119, CLC: CLF-1, NNT-1, BSF-3, CRLF1, CNTFRa, sorLA, Vps10p, STAT3, CILIARY NEUROTROPHIC FACTOR, COMPOSITE CYTOKINE, CYTOPLASMIC DOMAIN, MICE LACKING, FACTOR-I, RECEPTOR, PROTEIN, SORTILIN, FACTOR-1, BINDING",
author = "Larsen, {Jakob V.} and Petersen, {Claus M.}",
year = "2017",
month = "1",
doi = "10.3791/55019",
language = "English",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "Journal of Visualized Experiments",
number = "119",

}

RIS

TY - JOUR

T1 - SorLA and CLC

T2 - CLF-1-dependent Downregulation of CNTFR alpha as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

AU - Larsen, Jakob V.

AU - Petersen, Claus M.

PY - 2017/1

Y1 - 2017/1

N2 - The heterodimeric cytokine Cardiotrophin-like Cytokine: Cytokine-like Factor-1 (CLC: CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFR alpha to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-beta (gp130/LIFR beta) for signaling. Both CLC and CNTFR alpha are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFR alpha (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC: CLF-1, as well as the trimeric (CLC: CLF-1: CNTFR alpha) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC: CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFR alpha and sorLA, CNTFR alpha first binds CLC: CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFR alpha, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC: CLF-1. The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC: CLF-1-dependent downregulation of surface-membrane CNTFR alpha expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFR alpha; and iii) the lowered cellular response to CLC: CLF-1-stimulation upon sorLA-mediated downregulation of CNTFR alpha.

AB - The heterodimeric cytokine Cardiotrophin-like Cytokine: Cytokine-like Factor-1 (CLC: CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFR alpha to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-beta (gp130/LIFR beta) for signaling. Both CLC and CNTFR alpha are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFR alpha (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC: CLF-1, as well as the trimeric (CLC: CLF-1: CNTFR alpha) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC: CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFR alpha and sorLA, CNTFR alpha first binds CLC: CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFR alpha, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC: CLF-1. The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC: CLF-1-dependent downregulation of surface-membrane CNTFR alpha expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFR alpha; and iii) the lowered cellular response to CLC: CLF-1-stimulation upon sorLA-mediated downregulation of CNTFR alpha.

KW - Biochemistry

KW - Issue 119

KW - CLC: CLF-1

KW - NNT-1

KW - BSF-3

KW - CRLF1

KW - CNTFRa

KW - sorLA

KW - Vps10p

KW - STAT3

KW - CILIARY NEUROTROPHIC FACTOR

KW - COMPOSITE CYTOKINE

KW - CYTOPLASMIC DOMAIN

KW - MICE LACKING

KW - FACTOR-I

KW - RECEPTOR

KW - PROTEIN

KW - SORTILIN

KW - FACTOR-1

KW - BINDING

U2 - 10.3791/55019

DO - 10.3791/55019

M3 - Journal article

C2 - 28117780

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 119

ER -