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Role for a glycan phosphoinositol anchor in Fc gamma receptor synergy.

Green JM, Schreiber AD, Brown EJ - J. Cell Biol. (1997)

Bottom Line: Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding.While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB.These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
While many cell types express receptors for the Fc domain of IgG (Fc gamma R), only primate polymorphonuclear neutrophils (PMN) express an Fc gamma R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc gamma receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc gamma receptors. Jurkat T cells were stably transfected with cDNA encoding Fc gamma RIIA and/or Fc gamma RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either Fc gamma RIIA or Fc gamma RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fc gamma RIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca2+]i rise, as did crosslinking CD59 with Fc gamma RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc gamma receptors are not required for synergy. Replacement of the GPI anchor of Fc gamma RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc gamma RIIA cytoplasmic tail abolished synergy. While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB. These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.

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[Ca2+]i flux in cells expressing FcγRIIA containing the  ITAM mutation. Fura 2-AM preloaded J2Y→ F/3 cells were incubated with the mAbs IV.3 (anti-FcγRII) and 3G8 (anti-FcγRIII),  then analyzed by fluorimetry as described in Fig 2. The mAb  C305, specific for the TCR/CD3 complex, was added at 300 sec to  demonstrate that these cells are competent to flux [Ca2+]i.
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Figure 4: [Ca2+]i flux in cells expressing FcγRIIA containing the ITAM mutation. Fura 2-AM preloaded J2Y→ F/3 cells were incubated with the mAbs IV.3 (anti-FcγRII) and 3G8 (anti-FcγRIII), then analyzed by fluorimetry as described in Fig 2. The mAb C305, specific for the TCR/CD3 complex, was added at 300 sec to demonstrate that these cells are competent to flux [Ca2+]i.

Mentions: Activation of tyrosine phosphorylation and propagation of a tyrosine kinase cascade by receptor associated ITAMs is thought to be essential for Fcγ receptor signaling (16, 43). To determine whether this cascade had a role in Fcγ receptor synergy, Jurkat cells were transfected with FcγRIIIB and a mutant FcγRIIA in which tyrosines Y282 and Y298 contained within the ITAM were mutated to phenylalanines (J2Y→ F/3; Fig. 1, bottom). It has been shown in model systems that these tyrosines are required for [Ca2+]i flux when FcγRIIA is ligated alone (27, 28). No synergistic [Ca2+]i flux occurred in J2Y→ F/3 cells when FcγRIIA was ligated either alone or together with FcγRIIIB, although these cells were fully competent to increase [Ca2+]i in response to antigen receptor ligation (Fig. 4). Therefore, these tyrosines in the cytoplasmic tail of FcγRIIA are required for the synergistic [Ca2+]i rise. Thus both the GPI anchor of FcγRIIIB and the ITAM motif of FcγRIIA are required for synergy in calcium signaling.


Role for a glycan phosphoinositol anchor in Fc gamma receptor synergy.

Green JM, Schreiber AD, Brown EJ - J. Cell Biol. (1997)

[Ca2+]i flux in cells expressing FcγRIIA containing the  ITAM mutation. Fura 2-AM preloaded J2Y→ F/3 cells were incubated with the mAbs IV.3 (anti-FcγRII) and 3G8 (anti-FcγRIII),  then analyzed by fluorimetry as described in Fig 2. The mAb  C305, specific for the TCR/CD3 complex, was added at 300 sec to  demonstrate that these cells are competent to flux [Ca2+]i.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140207&req=5

Figure 4: [Ca2+]i flux in cells expressing FcγRIIA containing the ITAM mutation. Fura 2-AM preloaded J2Y→ F/3 cells were incubated with the mAbs IV.3 (anti-FcγRII) and 3G8 (anti-FcγRIII), then analyzed by fluorimetry as described in Fig 2. The mAb C305, specific for the TCR/CD3 complex, was added at 300 sec to demonstrate that these cells are competent to flux [Ca2+]i.
Mentions: Activation of tyrosine phosphorylation and propagation of a tyrosine kinase cascade by receptor associated ITAMs is thought to be essential for Fcγ receptor signaling (16, 43). To determine whether this cascade had a role in Fcγ receptor synergy, Jurkat cells were transfected with FcγRIIIB and a mutant FcγRIIA in which tyrosines Y282 and Y298 contained within the ITAM were mutated to phenylalanines (J2Y→ F/3; Fig. 1, bottom). It has been shown in model systems that these tyrosines are required for [Ca2+]i flux when FcγRIIA is ligated alone (27, 28). No synergistic [Ca2+]i flux occurred in J2Y→ F/3 cells when FcγRIIA was ligated either alone or together with FcγRIIIB, although these cells were fully competent to increase [Ca2+]i in response to antigen receptor ligation (Fig. 4). Therefore, these tyrosines in the cytoplasmic tail of FcγRIIA are required for the synergistic [Ca2+]i rise. Thus both the GPI anchor of FcγRIIIB and the ITAM motif of FcγRIIA are required for synergy in calcium signaling.

Bottom Line: Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding.While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB.These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
While many cell types express receptors for the Fc domain of IgG (Fc gamma R), only primate polymorphonuclear neutrophils (PMN) express an Fc gamma R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc gamma receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc gamma receptors. Jurkat T cells were stably transfected with cDNA encoding Fc gamma RIIA and/or Fc gamma RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either Fc gamma RIIA or Fc gamma RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fc gamma RIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca2+]i rise, as did crosslinking CD59 with Fc gamma RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc gamma receptors are not required for synergy. Replacement of the GPI anchor of Fc gamma RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc gamma RIIA cytoplasmic tail abolished synergy. While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB. These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.

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