Role for a Glycan Phosphoinositol Anchor in Fcγ Receptor Synergy

While many cell types express receptors for the Fc domain of IgG (FcγR), only primate polymorphonuclear neutrophils (PMN) express an FcγR linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked FcγR (FcγRIIIB) cooperates with the transmembrane FcγR (FcγRIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fcγ receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fcγ receptors. Jurkat T cells were stably transfected with cDNA encoding FcγRIIA and/or FcγRIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca(2+)](i)) to levels not reached by stimulation of either FcγRIIA or FcγRIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca(2+). Cocrosslinking FcγRIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca(2+)](i) rise, as did crosslinking CD59 with FcγRIIA on PMN, suggesting that interactions between the extracellular domains of the two Fcγ receptors are not required for synergy. Replacement of the GPI anchor of FcγRIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the FcγRIIA cytoplasmic tail abolished synergy. While the ITAM of FcγRIIA was required for the increase in [Ca(2+)](i), tyrosine phosphorylation of crosslinked FcγRIIA was diminished when cocrosslinked with FcγRIIIB. These data demonstrate that FcγRIIA association with GPI-linked proteins facilitates FcγR signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored FcγR of human PMN.