When a cell-surface receptor binds its ligand, the resulting complex is internalized into the cell. For a long time, this internalization was regarded as a mechanism for terminating signaling, leading to the degradation of the proteins involved. This view evolved with the discovery that certain receptors continue to emit signals from endosomal compartments after entering the cell. This concept of endosomal signaling, first described for receptor tyrosine kinases, was subsequently extended to the immune system, notably to the T-cell receptor (TCR). Given the strong similarity between the CD3ζ chain of the TCR and the γ chain of receptors for the Fc fragments of immunoglobulins (FcRs), the authors sought to determine whether these human Fc receptors could likewise exploit endosomal signaling platforms.
The study focused on three activating receptors: FcαRI, FcγRIIA, and FcγRI. After aggregating each one using specific antibodies, their intracellular trafficking was monitored by confocal microscopy, colocalization measurements, co-immunoprecipitation, and fluorescence lifetime imaging microscopy (FLIM). All three receptors are internalized once cross-linked, but their fates differ markedly. FcαRI is directed straight to lysosomes, a pathway associated with degradation. In contrast, FcγRIIA and FcγRI reach distinct endosomal compartments, identified by the presence of insulin-regulated aminopeptidase (IRAP), already recognized as a marker of TCR signaling endosomes. Within these compartments, the Fcγ receptors recruit active signaling molecules, including the active form of the kinase Syk, the phospholipase PLCγ, and the adaptor protein LAT.
To assess the functional importance of these platforms, the researchers studied cells lacking IRAP. The absence of this protein destabilizes the endosomal signaling of Fcγ receptors and leads to measurable functional consequences. Cytokine secretion triggered by FcγR activation, assessed by ELISA on peritoneal macrophages, is thereby impaired. Furthermore, the ability of macrophages to destroy tumor cells through antibody-dependent cellular cytotoxicity (ADCC) is also altered: in this assay, macrophages from wild-type or IRAP-deficient mice were exposed to tumor cells in the presence of increasing concentrations of an anti-EGFR antibody.
This work establishes that endosomal signaling is not merely a by-product of Fcγ receptor internalization, but a step required for their function. The authors conclude that this endosomal signaling is necessary for the FcγR-driven inflammatory response and, possibly, for the therapeutic action of monoclonal antibodies.