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Unravel mechanism of inside-out regulation of FcγRI

Thursday 3 February 2011

Arianne Brandsma and Dr. Jeanette Leusen

The concept of receptor activation by inside-out signaling has been well established for integrins and has also been documented for FcRI, FcRIIa and FcRI, representing an important step in FcR receptor functioning (Brandsma et al. Immunol Rev. 2015).

FcγRI is the only receptor with a high affinity for monomeric IgG. Because of its high affinity, FcγRI is believed to be saturated with circulating monomeric IgG and thus incapable of participating in antibody responses. However, in FcγRI knockout mice antibody-dependent cellular processes like bacterial clearance, phagocytosis, antigen presentation, and cytokine production are impaired, indicating that FcγRI is important for antibody responses.

FcγRI can be activated by cytokine-induced inside-out signaling, which can increase the binding capacity of FcγRI for immune complexes without changing surface receptor expression (Van der Poel et al., Blood 2010). However, the detailed mechanism(s) of FcγRI inside-out signaling are unknown. Interestingly, the increased binding capacity of FcγRI can be blocked by chemically inhibiting a serine/threonine phosphatase. This indicates that a dephosphorylation step is important for the activation of FcγRI, although phosphorylation of FcγRI itself not altered during inside-out signaling.

Figure 1. FcγRI structure within the plasma membrane. Indicated in red are three SNPs of FcγRI: in ectodomain 1, in the transmembrane region, and in the intracellular domain.
For integrins, a conformational change within the extracellular part of the ligand binding -chain has been documented. Whether FcγRI activation by cytokine-induced inside-out signaling also involves a conformational change in the extracellular domain contributing to increased immune complex binding is unknown. We are currently generating antibodies against a putative ‘active’ or ‘inactive’ conformation of FcγRI. The identification of such antibodies will provide evidence for a conformational change after FcγRI inside-out activation and they might be valuable tools in measuring the activation status of FcγRI on immune cells (e.g. of patients). In addition, fluorescent-resonance energy transfer (FRET) techniques in combination with different FcγRI-specific antibodies can be used to measure the conformational changes of FcγRI after inside-out signaling.

Several single nucleotide polymorphisms (SNPs) have been identified for FcγRI (Van der Poel, JI 2011). We are currently investigating the functional consequences of these SNPs, also in the context of inside-out signaling.

In this project, the newly generated antibodies against FcγRI will be characterized in vitro using flow cytometry and ELISA. In addition, the role of the SNPs of FcγRI on ligand binding and inside-out signaling will be studied. Finally, using cell biological techniques the mechanism of the inside-out signaling of FcγRI will be studied in more detail.

Molecular biology, site-directed mutagenesis, transfections, cell culture, isolation of leukocytes from human blood, FACS, ELISA, Mouse models, Western blot, microscopy

6 or 9 months

Dr. Jeanette Leusen, jleusen@umcutrecht.nl
Dr. Kristin Denzer, k.denzer@umcutrecht.nl, 088 75 643 68

A.M. Brandsma, S.R. Jacobino, S. Meyer, T. ten Broeke, and J.H.W. Leusen. Fc receptor inside out signaling and possible impact on antibody therapy. Immunol Rev. 2015;268:74-87.

C.E. van der Poel, R.A. Karssemeijer, P. Boross, J.A. van der Linden, M. Blokland, J.G.J. van de Winkel, and J.H.W. Leusen. Cytokine-induced immune complex binding to the high-affinity IgG receptor, FcgammaRI, in the presence of monomeric IgG. Blood 2010;116:5327-5333.

C.E. van der Poel, R.M. Spaapen, J.G.J. van de Winkel and J.H.W. Leusen. Functional characteristics of the high affinity IgG receptor, FcgammaRI. J. Immunol 2011;186:2699-2704.