Serine phosphorylation of FcγRI cytoplasmic domain directs lipid raft localization and interaction with protein 4.1G

The high-affinity IgG receptor (CD64, Fc gamma RI) 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 Fc gamma RI interacts with protein 4.1G (EPB41L2). Our mutational analyses identified two required 4.1G-interacting regions in the Fc gamma RI CY and one Fc gamma RI-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 Fc gamma RI CY and influence Fc gamma RI membrane mobility and function. We show that Fc gamma RI CY interacts with protein 4.1G in vitro and that Fc gamma RI co-immunoprecipitates protein 4.1G in freshly isolated human PBMC. With the use of immunostaining, we show that Fc gamma RI colocalizes with protein 4.1G in unstimulated U937 cells, in which the Fc gamma RI CY is constitutively serine-phosphorylated, but significant uncoupling occurs following Fc gamma RI cross-linking, suggesting phosphoserine-regulated interaction. In vitro, protein 4.1G interacted preferentially with CK2-phosphorylated Fc gamma RI CY, and compared with WT Fc gamma RI, a nonphosphorylatable Fc gamma RI mutant receptor was excluded from lipid rafts, suggesting a key role for protein 4.1G in targeting phosphorylated Fc gamma RI to rafts. These data are consistent with a phosphoserine-dependent tethering role for protein 4.1G in maintaining Fc gamma RI in lipid rafts and provide insight into the unique phosphoserine-based regulation of receptor signaling by Fc gamma RI CY. J. Leukoc. Biol. 91: 97-103; 2012.