The Fc receptor supramolecular complex from RAW macrophages
thesisposted on 2022-10-05, 17:40 authored by Veronica Erma Barbisan
The Fc receptor is expressed on the surface of RAW macrophages that can engulf IgG opsonized particles by phagocytosis. Isolation and characterization of these receptors and the identification of proteins involved is challenging. Live-cell Affinity Receptor Chromatography (LARC) (Janowski 2008) resulted in the identification of 336 proteins which are found to be specifically associated with the activated Fc receptor in RAW 264.7 macrophage cells. The proteins isolated were analyzed for protein-protein interactions using various analytical techniques including data mining through the use of iHOP, protein interaction databases such as Cytoscape, Osprey and STRING and literature searches, in order to create a signalling interaction pathway involving the proteins that are activated and associated with the Fcy receptor during receptor-mediated phagocytosis. The phospholipase C family of proteins have been previously identified as important in phagocytosis, and were identified by LARC. The selected isoforms were confirmed in RAW 264.7 cells via Western Blots, antibody staining, and live-cell confocal microscopy with fluorescently linked fusion proteins to verify the localization of the phospholipases, specifically PLC-β₃, PLC-β₄, PLC-y₁, PLC-d₁, PLC-ε₁ and PLC like -2 to the activated receptor complex.