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The common FcR?, an immunoreceptor tyrosine-based activation motif (ITAM)- containing adaptor protein, associates with multiple leukocyte receptor complexes and mediates signal transduction through the ITAM in the cytoplasmic domain. The presence of multiple serine and threonine residues within this motif suggests the potential for serine/threonine phosphorylation in modulating signaling events. Single-site mutational analysis of these residues in RBL-2H3 cells indicates that each may contribute to net FcR?-mediated signaling, and mass spectrometry of WT human FcR? from receptor-stimulated cells shows consistent preferential phosphorylation of the serine residue at position 51. Immunoblot analysis, mass spectrometry, and mutational analyses showed that phosphorylation of serine 51 in the 7-residue spacer between the 2 YxxL sequences regulates FcR? signaling by inhibiting tyrosine phosphorylation at the membrane proximal Y47 position of the ITAM, but not phosphorylation at position Y58. This inhibition results in reduced Syk recruitment and activation. With in vitro kinase assays, PKC-d and PKA show preferential phosphorylation of S51. Serine/threonine phosphorylation of the FcR? ITAM, which functions as an integrator of multiple signaling elements, may explain in part the contribution of variants in PKC-d and other PKC isoforms to some autoimmune phenotypes.

Serine phosphorylation of the FcγRI CY domain directs its migration to lipid rafts and its interaction with protein 4.1G, suggesting a tethering role for protein 4.1G. The high‐affinity IgG receptor (CD64, FcγRI) has several special capacities, including the receptor‐stimulated cleavage of the cell surface B cell‐activating factor of the TNF superfamily (TNFSF13B). With the use of the yeast two‐hybrid system, we and others have shown that FcγRI interacts with protein 4.1G (EPB41L2). Our mutational analyses identified two required 4.1G‐interacting regions in the FcγRI CY and one FcγRI‐interacting site in the C‐terminus of protein 4.1G. Herein, we explore mechanism(s) that may regulate the interaction between protein 4.1G and FcγRI CY and influence FcγRI membrane mobility and function. We show that FcγRI CY interacts with protein 4.1G in vitro and that FcγRI coimmunoprecipitates protein 4.1G in freshly isolated human PBMC. With the use of immunostaining, we show that FcγRI colocalizes with protein 4.1G in unstimulated U937 cells, in which the FcγRI CY is constitutively serine‐phosphorylated, but significant uncoupling occurs following FcγRI cross‐linking, suggesting phosphoserine‐regulated interaction. In vitro, protein 4.1G interacted preferentially with CK2‐phosphorylated FcγRI CY, and compared with WT FcγRI, a nonphosphorylatable FcγRI mutant receptor was excluded from lipid rafts, suggesting a key role for protein 4.1G in targeting phosphorylated FcγRI to rafts. These data are consistent with a phosphoserine‐dependent tethering role for protein 4.1G in maintaining FcγRI in lipid rafts and provide insight into the unique phosphoserine‐based regulation of receptor signaling by FcγRI CY.

FcαRI (CD89) is a human IgA FcR expressed on cells of myeloid lineage such as neutrophils, monocytes, tissue macrophages, eosinophils, and subpopulations of dendritic cells. FcαRI mediates cell activation through Src family kinases and downstream tyrosine‐based phosphorylation pathways. However, the role of IgA and the expression and role of its cognate receptor FcαRI (CD89) in platelet activation are undefined. In the current study, we demonstrate that human platelets express FcαRI mRNAs and proteins. Furthermore, we show that the platelet FcαRI is associated with the FcR γ‐chain, and cross‐linking of FcαRI leads to Syk phosphorylation. Clustering of FcαRI induces pre‐mRNA splicing and protein production of tissue factor and IL‐1β, suggesting novel roles for human platelet FcαRI and serum IgA in thrombosis and inflammation.