Biophysical and biochemical techniques for the analysis and targeting of the Fc receptor supramolecular complex

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We will analyze a model receptor, the Fc receptor, by mass spectrometry and live-cell microscopy over the time course of IgG-microbead binding, recognition and activation. Receptors are proteins expressed on the surface of cells that control the responses to messengers circulating in the body and are critical targets in the treatment of disease and pain. There is a great need to understand and compare receptors on the surface of cells and to create new and specific drugs against them. Many responses to tissue injury such as wounds and infection involve the activation of special cells called macrophages that in turn trigger an inflammatory response. In most instances, inflammation is beneficial, but occasionally an aberrant inflammatory response can initiate a disease process, e.g., arthritic inflammation of the joints, atherosclerotic inflammation of the arteries, or like cancer and Alzheimer's dementia. Human immunoglobulin G (IgG) or its fragment crystalizable (Fc) that flags invading bacteria for destruction may be used to capture their receptors and/or co-receptors from macrophage cell lines including human differentiated U937 cells and RAW macrophages. We aim to activate and then capture the non-inflammatory, monovalent Fc gamma receptor-IgG-S-S-biotin complex for collection over strepavidin, and release by a simple reduction in one step. Aggregated IgG-microbeads will activate and capture the activated phagocytic complex directly from the surface of living cells for isolation and characterization by mass spectrometry. Mass spectrometry, biochemical assays and live-cell laser microscopy of the ligand coated microbeads with cultured macrophages will reveal and confirm the mechanisms of Fc activation that regulate phagocytosis and free radical production. We observed the Fc gamma, epsilon, alpha, mu receptors were tightly associated with specific isoforms of Fc-like receptors (FCLR), Leukocyte Immunoglobulin-Like receptors (LILR), killer cell (KIR) and Killer lectin receptors (KLR) that were present together in a supramolecular complex on IgG-, but not oxLDL or anti CD36, microbeads. Silencing RNA against the specifically discovered complex of Fc receptors and co-receptors will demonstrate the role of the co-receptors in phagocytosis or free radical production. Macrophages and neutrophils are phagocytes and thus possess the capacity to release free radicals like superoxide (O2-*) that causes the formation of H2O2 during the engulfment and destruction of IgG-bacteria complexes. However, there are few absolute quantitative methods available for measuring the low amounts of H2O2 products from most cell types to date. We have shown that a standard curve and H2O2 assay can be created in cell growth media and will detect H2O2 leaving the cell down to approximately 1 pico mol levels in response to IgG and other ligands, drugs or receptor siRNA. The Fc receptor CD64 will be expressed on the surface of naive cells that will be fixed and used as a chromatography column to capture the receptor ligand IgG or Fc peptide (verses the control naive cells). A number of receptors for epidermal growth factors (EGF), interleukins (IL) and colony stimulating factors (CSF) and FLT3R were found with the IgG-Fc receptor complex. The FLT3 ligand was found to regulate the phagocytosis of IgG-coated microbeads and we will test the effect of the implicated in phagocytosis and Hydrogen peroxide production of IgG-coated microbeads. We will provide a new and powerful way to capture cell surface receptors and co-receptors, measure their roles in free radical release and phagocytosis, and then rapidly screen and test their protein ligands that might serve as biological drugs targeted against the receptor-specific inflammatory functions.