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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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The synergistic rise in [Ca2+]i requires the influx of extracellular calcium. Changes in Fura 2-AM fluorescence after receptor  crosslinking in J2/3 cells was measured as in Fig. 2 in the absence or presence of 2 mM EGTA to prevent calcium influx from the medium. (A) 2 mM EGTA was added 280 s after crosslinking. (B) 2 mM EGTA was added immediately before receptor crosslinking. Also  shown is no added EGTA. (C) 2 mM EGTA was added at 0 or 300 s.
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Figure 7: The synergistic rise in [Ca2+]i requires the influx of extracellular calcium. Changes in Fura 2-AM fluorescence after receptor crosslinking in J2/3 cells was measured as in Fig. 2 in the absence or presence of 2 mM EGTA to prevent calcium influx from the medium. (A) 2 mM EGTA was added 280 s after crosslinking. (B) 2 mM EGTA was added immediately before receptor crosslinking. Also shown is no added EGTA. (C) 2 mM EGTA was added at 0 or 300 s.

Mentions: To determine the source of Ca2+ for the synergistic [Ca2+]i rise in the J2/3 cells, changes in Fura-2 fluorescence were measured in the presence of extracellular EGTA to prevent calcium influx from the medium. The synergistic [Ca2+]i rise was inhibited almost immediately after addition of EGTA, indicating that calcium influx through plasma membrane channels is largely responsible for the prolonged [Ca2+]i rise (Fig. 7 A, left) as found in PMN (44). Similarly, the synergistic [Ca2+]i rise induced by cocrosslinking FcγRIIA and CD59 was abolished by the addition of EGTA (Fig. 7 A, middle). As a control, the changes in intracellular calcium were measured after the T-cell receptor complex (TCR/CD3) was crosslinked with the mAb C305 (Fig. 7 A, right). Previous studies have shown that the rise in intracellular calcium after TCR crosslinking results from an initial rise derived from intracellular stores followed by a secondary sustained calcium influx through plasma membrane channels that can be abolished by the addition of EGTA (41). The addition of EGTA to Jurkat cells treated only with crosslinking secondary antibody does cause a small decrease in the amount of intracellular calcium, but this small depletion does not account for the large loss in the synergistic calcium influx from extracellular stores, as previously shown in PMN (37; Fig. 7, A and C, left). The changes in intracellular calcium also were measured when EGTA was added immediately before Fcγ receptor crosslinking (Fig. 7 B, left). Crosslinking led to an initial rise in [Ca2+]i, but the synergistic [Ca2+]i rise was substantially diminished after cocrosslinking FcγRIIA with FcγRIIIB or CD59 (Fig. 7 B, middle and right). The magnitude of the [Ca2+]i rise also was diminished in the presence of EGTA, again demonstrating that a significant contribution to the [Ca2+]i rise is due to the influx of extracellular calcium (Fig. 7 B). The slow rise in [Ca2+]i after crosslinking either FcγRIIIB or CD59 alone was abolished in the presence of EGTA (Fig. 7 C, right, and data not shown). EGTA treated cells do not produce a flux in [Ca2+]i after the addition of crosslinking secondary antibodies alone (Fig. 7 C, left).


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

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

The synergistic rise in [Ca2+]i requires the influx of extracellular calcium. Changes in Fura 2-AM fluorescence after receptor  crosslinking in J2/3 cells was measured as in Fig. 2 in the absence or presence of 2 mM EGTA to prevent calcium influx from the medium. (A) 2 mM EGTA was added 280 s after crosslinking. (B) 2 mM EGTA was added immediately before receptor crosslinking. Also  shown is no added EGTA. (C) 2 mM EGTA was added at 0 or 300 s.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: The synergistic rise in [Ca2+]i requires the influx of extracellular calcium. Changes in Fura 2-AM fluorescence after receptor crosslinking in J2/3 cells was measured as in Fig. 2 in the absence or presence of 2 mM EGTA to prevent calcium influx from the medium. (A) 2 mM EGTA was added 280 s after crosslinking. (B) 2 mM EGTA was added immediately before receptor crosslinking. Also shown is no added EGTA. (C) 2 mM EGTA was added at 0 or 300 s.
Mentions: To determine the source of Ca2+ for the synergistic [Ca2+]i rise in the J2/3 cells, changes in Fura-2 fluorescence were measured in the presence of extracellular EGTA to prevent calcium influx from the medium. The synergistic [Ca2+]i rise was inhibited almost immediately after addition of EGTA, indicating that calcium influx through plasma membrane channels is largely responsible for the prolonged [Ca2+]i rise (Fig. 7 A, left) as found in PMN (44). Similarly, the synergistic [Ca2+]i rise induced by cocrosslinking FcγRIIA and CD59 was abolished by the addition of EGTA (Fig. 7 A, middle). As a control, the changes in intracellular calcium were measured after the T-cell receptor complex (TCR/CD3) was crosslinked with the mAb C305 (Fig. 7 A, right). Previous studies have shown that the rise in intracellular calcium after TCR crosslinking results from an initial rise derived from intracellular stores followed by a secondary sustained calcium influx through plasma membrane channels that can be abolished by the addition of EGTA (41). The addition of EGTA to Jurkat cells treated only with crosslinking secondary antibody does cause a small decrease in the amount of intracellular calcium, but this small depletion does not account for the large loss in the synergistic calcium influx from extracellular stores, as previously shown in PMN (37; Fig. 7, A and C, left). The changes in intracellular calcium also were measured when EGTA was added immediately before Fcγ receptor crosslinking (Fig. 7 B, left). Crosslinking led to an initial rise in [Ca2+]i, but the synergistic [Ca2+]i rise was substantially diminished after cocrosslinking FcγRIIA with FcγRIIIB or CD59 (Fig. 7 B, middle and right). The magnitude of the [Ca2+]i rise also was diminished in the presence of EGTA, again demonstrating that a significant contribution to the [Ca2+]i rise is due to the influx of extracellular calcium (Fig. 7 B). The slow rise in [Ca2+]i after crosslinking either FcγRIIIB or CD59 alone was abolished in the presence of EGTA (Fig. 7 C, right, and data not shown). EGTA treated cells do not produce a flux in [Ca2+]i after the addition of crosslinking secondary antibodies alone (Fig. 7 C, left).

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