title: EGF Uptake and Degradation Assay to Determine the Effect of HTLV Regulatory Proteins on the ESCRT-Dependent MVB Pathway.
abstract: The endosomal sorting complex required for transport (ESCRT) pathway plays key roles in multivesicular bodies (MVBs) formation and lysosomal degradation of membrane receptors, viral budding, and midbody abscission during cytokinesis. The epidermal growth factor receptor (EGFR) is regarded as a prototypical cargo of the MVB/ESCRT pathway and following stimulation by epidermal growth factor (EGF) EGFR/EGF complexes are internalized, sorted into MVBs, and degraded by lysosomes or recycled back to the cell membrane. Here, we describe an assay to analyze the effect of human T-cell leukemia (HTLV) regulatory proteins on the functionality of ESCRT-dependent MVB/lysosomal trafficking of EGFR/EGF complexes. This is performed by direct visualization and quantification of the rate of EGF-Alexa595/EGFR internalization and degradation in HeLa cells expressing HTLV regulatory proteins by immunofluorescence and western blot.
authors.affiliation: UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Colin
authors.initials: C
authors.lastname: Murphy
authors.affiliation: Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland. Noreen.sheehy@ucd.ie.
authors.forename: Noreen
authors.initials: N
authors.lastname: Sheehy
mesh_terms: D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D014764:Viral Proteins; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: APH-2; EGF; ESCRT; HBZ; HTLV; MVBs; Tax1