FcepsilonRI ubiquitylation negatively regulates receptor tyrosine phosphorylation and mast cell effector function

The high-affinity IgE receptor, FcepsilonRI is a tetrameric immune receptor expressed on mast cells. Upon receptor aggregation, FcepsilonRI beta and gamma subunits are phosphorylated and ubiquitylated. Receptor phosphorylation functions to recruit and activate signaling complexes leading to the activation of effector responses. Ubiquitylation of components of signaling complexes negatively regulates cell activation; however, the specific function of FcepsilonRI ubiquitylation has not been characterized. Whether or not ubiquitylation of immune receptors regulates cell activation, as has been demonstrated for receptor tyrosine kinases, is unknown.; To specifically analyze the function of FcepsilonRI ubiquitylation, we engineered lysine-to-argine amino-acid substitutions in the cytoplasmic domains of FcepsilonRI gamma and beta subunits, to block the capacity of the proteins to be ubiquitylated. Ubiquitylation-incompetent FcepsilonRI subunits were expressed in U937 cells. K-R mutant FcepsilonRI are expressed on the cell surface at levels similar to wild-type FcepsilonRl and can function to activate signals leading to calcium mobilization. K-R substitution abrogates receptor ubiquitylation. K-R substitution mediates enhanced FcepsilonRI tyrosine phosphorylation upon receptor aggregation, but does not mediate enhanced calcium mobilization.; To further characterize the function of FcepsilonRI ubiquitylation. K-R mutant FcRbeta and FcRgamma components were expressed in FCRbeta-deficient and FcRgamma-deficient BMMC respectively. Consistent with the U937 model, K-R FcRbeta mediates enhanced FcepsilonRI tyrosine phosphorylation. K-R FcRbeta also mediates enhanced cytokine release and degranulation. By contrast, K-R mutant FcRgamma mediates impaired effector responses. We suggest that K-R substitution in FcRgamma impairs the ability of PKCdelta to phosphorylate a threonine residue in the FcRgamma ITAM. This modification, in conjunction with tyrosine phosphorylation, is required for syk activation. To bypass this requirement, we substituted the ITAM threonine residue with an aspartate residue to mimic the acidic phospho-threonine. This substitution was introduced to both the K-R and wild-type FcRgamma proteins. K-R,T-D FcRgamma mediates enhanced FcepsilonRI tyrosine phosphorylation, cytokine release and degranulation, similar to that observed with the K-R FcRbeta protein. In both the U937 and BMMC models, K-R substitution did not change the rate of receptor endocytosis.; These data are consistent with the hypothesis that ubiquitylation of components of immune receptors can negatively regulate receptor phosphorylation and down-stream effector responses.