Role for a Glycan Phosphoinositol Anchor in Fc g Receptor Synergy

While many cell types express receptors for the Fc domain of IgG (Fc g R), only primate polymorphonuclear neutrophils (PMN) express an Fc g R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fc g R (Fc g RIIIB) cooperates with the transmembrane Fc g R (Fc g RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc g receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc g receptors. Jurkat T cells were stably transfected with cDNA encoding Fc g RIIA and/or Fc g RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca 2 1 ] i ) to levels not reached by stimulation of either Fc g RIIA or Fc g RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca 2 1 . Cocrosslinking Fc g RIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca 2 1 ] i rise, as did crosslinking CD59 with Fc g RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc g receptors are not required for synergy. Replacement of the GPI anchor of Fc g RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosinebased activation motif (ITAM) of the Fc g RIIA cytoplasmic tail abolished synergy. While the ITAM of Fc g RIIA was required for the increase in [Ca 2 1 ] i , tyrosine phosphorylation of crosslinked Fc g RIIA was diminished when cocrosslinked with Fc g RIIIB. These data demonstrate that Fc g RIIA association with GPI-linked proteins facilitates Fc g R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc g R of hu-