Ligand specificity characterizes receptors for Abs and many other immune receptors, but the common use of the FcR γ-chain as their signaling subunit challenges the concept that these receptors are functionally distinct. We hypothesized that elements for specificity might be determined by the unique cytoplasmic domain (CY) sequences of the ligand-binding α-chains of γ-chain-associated receptors. Among Fcγ receptors, a protein kinase C (PKC) phosphorylation consensus motif [RSSTR], identified within the FcγRIIIa (CD16A) CY by in silico analysis, is specifically phosphorylated by PKCs, unlike other FcRs. Phosphorylated CD16A mediates a more robust calcium flux, tyrosine phosphorylation of Syk, and proinflammatory cytokine production, whereas non-phosphorylatable CD16A is more effective at activation of the Gab2/PI3K pathway, leading to enhanced degranulation. S100A4, a specific protein-binding partner for CD16A-CY newly identified by yeast two-hybrid analysis, inhibits phosphorylation of CD16A-CY by PKC in vitro, and reduction of S100A4 levels in vivo enhances receptor phosphorylation upon cross-linking. Taken together, PKC-mediated phosphorylation of CD16A modulates distinct signaling pathways engaged by the receptor. Calcium-activated binding of S100A4 to CD16A, promoted by the initial calcium flux, attenuates the phosphorylation of CY, and, acting as a molecular switch, may both serve as a negative feedback on cytokine production pathways during sustained receptor engagement and favor a shift to degranulation, consistent with the importance of granule release following conjugate formation between CD16A+ effector cells and target cells. This switch mechanism points to new therapeutic targets and provides a framework for understanding novel receptor polymorphisms. Copyright © 2012 by The American Association of Immunologists, Inc.