title: SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα 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 CNTFRα to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-β (gp130/LIFRβ) for signaling. Both CLC and CNTFRα 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 CNTFRα (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:CNTFRα) 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 CNTFRα and sorLA, CNTFRα 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, CNTFRα, 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 CNTFRα expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFRα; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFRα.
authors.affiliation: Department of Biomedicine, Aarhus University; jvl@biomed.au.dk.
authors.forename: Jakob V
authors.initials: JV
authors.lastname: Larsen
authors.affiliation: Department of Biomedicine, Aarhus University; cmp@biomed.au.dk.
authors.forename: Claus M
authors.initials: CM
authors.lastname: Petersen
mesh_terms: D001665:Binding Sites; D015153:Blotting, Western; D053739:Ciliary Neurotrophic Factor Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D026502:LDL-Receptor Related Proteins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D018121:Receptors, Cytokine / Q000737:chemistry / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D053739:Ciliary Neurotrophic Factor Receptor alpha Subunit; D016207:Cytokines; D026502:LDL-Receptor Related Proteins; D008024:Ligands; D051907:Lysosomal Membrane Proteins; D026901:Membrane Transport Proteins; D018121:Receptors, Cytokine; D050796:STAT3 Transcription Factor; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C102582:SORL1 protein, human; C121169:cardiotrophin-like cytokine; C113554:cytokine-like factor-1