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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

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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.

TidsskriftJournal of Visualized Experiments
Antal sider7
StatusUdgivet - jan. 2017

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