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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 in cells expressing the chimeric FcγRIIIB/CD7.  J2/3 cells (top), J2/3-CD7 cells (middle), or PMN (bottom) were  preloaded with Fura 2-AM. J2/3 and J2/3-CD7 cells were then incubated for 30 min with the mAb IV.3 (anti-FcγRII), mAb 3G8  (anti-FcγRIII), mAb MEM-43 (anti-CD59), or combinations of  these mAbs. PMN were incubated with mAb IV.3 F(ab), mAb  10G10 F(ab′)2 (anti-CD59), or combinations of these mAbs. Experiments were performed as described in Fig. 2. Each curve is  representative of at least three independent experiments. For  PMN, the change in [Ca2+]i at 140 s after the addition of crosslinking antibody was calculated and results are shown as the  mean ± SEM for three independent experiments (bottom).
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Figure 3: [Ca2+]i in cells expressing the chimeric FcγRIIIB/CD7. J2/3 cells (top), J2/3-CD7 cells (middle), or PMN (bottom) were preloaded with Fura 2-AM. J2/3 and J2/3-CD7 cells were then incubated for 30 min with the mAb IV.3 (anti-FcγRII), mAb 3G8 (anti-FcγRIII), mAb MEM-43 (anti-CD59), or combinations of these mAbs. PMN were incubated with mAb IV.3 F(ab), mAb 10G10 F(ab′)2 (anti-CD59), or combinations of these mAbs. Experiments were performed as described in Fig. 2. Each curve is representative of at least three independent experiments. For PMN, the change in [Ca2+]i at 140 s after the addition of crosslinking antibody was calculated and results are shown as the mean ± SEM for three independent experiments (bottom).

Mentions: Primate PMN are the only cells that express a GPI-anchored Fcγ receptor (32). To determine whether the GPI anchor was necessary for FcγRIIIB contribution to the synergistic increase in [Ca2+]i, stable transfectants were made expressing FcγRIIA and a chimeric FcγRIIIB with the GPI anchor replaced by the transmembrane domain of CD7 (J2/3-CD7; Fig. 1, middle). When FcγRIIA and FcγRIIIB/ CD7 were crosslinked together in these cells, the [Ca2+]i rise was similar to the rise generated when FcγRIIA was crosslinked alone without any synergy from FcγRIIIB (Fig. 3, middle). The inability of the chimeric FcγRIIIB/ CD7 molecule to contribute to the synergistic [Ca2+]i rise was not due to inadequate expression of this protein, since the FcγRIIIB/CD7 molecule was expressed at a greater level than the wild-type FcγRIIIB (Fig. 1, top and middle). This experiment demonstrates that the GPI anchor is necessary for the synergistic [Ca2+]i rise.


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

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

[Ca2+]i in cells expressing the chimeric FcγRIIIB/CD7.  J2/3 cells (top), J2/3-CD7 cells (middle), or PMN (bottom) were  preloaded with Fura 2-AM. J2/3 and J2/3-CD7 cells were then incubated for 30 min with the mAb IV.3 (anti-FcγRII), mAb 3G8  (anti-FcγRIII), mAb MEM-43 (anti-CD59), or combinations of  these mAbs. PMN were incubated with mAb IV.3 F(ab), mAb  10G10 F(ab′)2 (anti-CD59), or combinations of these mAbs. Experiments were performed as described in Fig. 2. Each curve is  representative of at least three independent experiments. For  PMN, the change in [Ca2+]i at 140 s after the addition of crosslinking antibody was calculated and results are shown as the  mean ± SEM for three independent experiments (bottom).
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Figure 3: [Ca2+]i in cells expressing the chimeric FcγRIIIB/CD7. J2/3 cells (top), J2/3-CD7 cells (middle), or PMN (bottom) were preloaded with Fura 2-AM. J2/3 and J2/3-CD7 cells were then incubated for 30 min with the mAb IV.3 (anti-FcγRII), mAb 3G8 (anti-FcγRIII), mAb MEM-43 (anti-CD59), or combinations of these mAbs. PMN were incubated with mAb IV.3 F(ab), mAb 10G10 F(ab′)2 (anti-CD59), or combinations of these mAbs. Experiments were performed as described in Fig. 2. Each curve is representative of at least three independent experiments. For PMN, the change in [Ca2+]i at 140 s after the addition of crosslinking antibody was calculated and results are shown as the mean ± SEM for three independent experiments (bottom).
Mentions: Primate PMN are the only cells that express a GPI-anchored Fcγ receptor (32). To determine whether the GPI anchor was necessary for FcγRIIIB contribution to the synergistic increase in [Ca2+]i, stable transfectants were made expressing FcγRIIA and a chimeric FcγRIIIB with the GPI anchor replaced by the transmembrane domain of CD7 (J2/3-CD7; Fig. 1, middle). When FcγRIIA and FcγRIIIB/ CD7 were crosslinked together in these cells, the [Ca2+]i rise was similar to the rise generated when FcγRIIA was crosslinked alone without any synergy from FcγRIIIB (Fig. 3, middle). The inability of the chimeric FcγRIIIB/ CD7 molecule to contribute to the synergistic [Ca2+]i rise was not due to inadequate expression of this protein, since the FcγRIIIB/CD7 molecule was expressed at a greater level than the wild-type FcγRIIIB (Fig. 1, top and middle). This experiment demonstrates that the GPI anchor is necessary for the synergistic [Ca2+]i rise.

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.

Show MeSH
Related in: MedlinePlus