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PAG3/Papalpha/KIAA0400, a GTPase-activating protein for ADP-ribosylation factor (ARF), regulates ARF6 in Fcgamma receptor-mediated phagocytosis of macrophages.

Uchida H, Kondo A, Yoshimura Y, Mazaki Y, Sabe H - J. Exp. Med. (2001)

Bottom Line: Overexpression of PAG3, but not its GAP-inactive mutant, attenuated the focal accumulation of F-actin and blocked phagocytosis, although surface levels of the FcgammaRs were not affected.Moreover, cooverexpression of ARF6, but not ARF1 or ARF5, restored the phagocytic activity of PAG3-overexpressing cells.We propose that PAG3 acts as a GAP for ARF6 and is hence involved in FcgammaR-mediated phagocytosis in mouse macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan.

ABSTRACT
The Fcgamma receptor (FcgammaR)-mediated phagocytosis of macrophages is a complex process where remodeling of both the actin-based cytoskeleton and plasma membrane occur coordinately. Several different families of small GTPases are involved. We have isolated a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF), paxillin-associated protein with ARFGAP activity (PAG)3/Papalpha/KIAA0400, from mature monocytes and macrophage-like cells. Mammalian ARFs fall into three classes, and the class III isoform (ARF6) has been shown to be involved in FcgammaR-mediated phagocytosis. Here we report that PAG3 is enriched together with ARF6 and F-actin at phagocytic cups formed beneath immunoglobulin G-opsonized beads in P388D1 macrophages, in which overexpression of ARF6, but not ARF1 (class I) or ARF5 (class II), inhibits the phagocytosis. Overexpression of PAG3, but not its GAP-inactive mutant, attenuated the focal accumulation of F-actin and blocked phagocytosis, although surface levels of the FcgammaRs were not affected. Other ubiquitously expressed ARFGAPs, G protein-coupled receptor kinase interactors GIT2 and GIT2-short/KIAA0148, which we have shown to exhibit GAP activity for ARF1 in COS-7 cells, did not accumulate at the phagocytic cups or inhibit phagocytosis. Moreover, cooverexpression of ARF6, but not ARF1 or ARF5, restored the phagocytic activity of PAG3-overexpressing cells. We propose that PAG3 acts as a GAP for ARF6 and is hence involved in FcgammaR-mediated phagocytosis in mouse macrophages.

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Cooverexpression of ARF6, but not other classes of ARF isoforms, restores the PAG3-induced inhibition of FcγR-mediated phagocytosis. P388D1 cells and cells transiently transfected with the indicated plasmid constructs in pEGFP and pcDNA3 vectors for overexpression were incubated with IgG-opsonized beads, and the phagocytosis index was determined, as described in the legend to Fig. 2. Plasmid DNAs for EGFP-PAG3 (WT) or its GAP-inactive mutant (CA) were cotransfected with plasmid DNAs for ARF1-HA (A), ARF5-HA (B), and ARF6-HA (C). In A–C, 2.0 μg of plasmid DNA for EGFP-PAG3 (bar 2), 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for each ARF-HA (bar 3), and 2.0 μg of plasmid DNA for each ARF-HA (bar 4) were used; the GAP-inactive mutant of EGFP-PAG3 was used instead of wild-type EGFP-PAG3 in bars 5–7, as indicated. In bars 8 and 9 in C, 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for the Q67L mutant (bar 8) or the T27N mutant (bar 9) of ARF6-HA were used. Mock-transfected controls were also included (bar 1 in A–C). The overexpression of exogenous proteins were confirmed as described in the legend to Fig. 2 and Fig. 3, with each cell examined for the phagocytic activities. In bar 3 in C, these cells expressed EGFP-PAG3 at levels 25–30-fold higher than endogenous PAG3, and expressed ARF6-HA at levels 7–9-fold higher than endogenous ARF6 (also see Fig. 4 B). In bars 2 and 4 in C, the expression levels of EGFP-PAG3 and ARF6-HA were essentially the same as those described in the legend to Fig. 2 and Fig. 3. Expression of ARF1-HA and ARF5-HA in A and B was also similar to those in Fig. 2. Data are expressed as the mean ± SE of three independent experiments. *P < 0.001.
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Figure 5: Cooverexpression of ARF6, but not other classes of ARF isoforms, restores the PAG3-induced inhibition of FcγR-mediated phagocytosis. P388D1 cells and cells transiently transfected with the indicated plasmid constructs in pEGFP and pcDNA3 vectors for overexpression were incubated with IgG-opsonized beads, and the phagocytosis index was determined, as described in the legend to Fig. 2. Plasmid DNAs for EGFP-PAG3 (WT) or its GAP-inactive mutant (CA) were cotransfected with plasmid DNAs for ARF1-HA (A), ARF5-HA (B), and ARF6-HA (C). In A–C, 2.0 μg of plasmid DNA for EGFP-PAG3 (bar 2), 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for each ARF-HA (bar 3), and 2.0 μg of plasmid DNA for each ARF-HA (bar 4) were used; the GAP-inactive mutant of EGFP-PAG3 was used instead of wild-type EGFP-PAG3 in bars 5–7, as indicated. In bars 8 and 9 in C, 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for the Q67L mutant (bar 8) or the T27N mutant (bar 9) of ARF6-HA were used. Mock-transfected controls were also included (bar 1 in A–C). The overexpression of exogenous proteins were confirmed as described in the legend to Fig. 2 and Fig. 3, with each cell examined for the phagocytic activities. In bar 3 in C, these cells expressed EGFP-PAG3 at levels 25–30-fold higher than endogenous PAG3, and expressed ARF6-HA at levels 7–9-fold higher than endogenous ARF6 (also see Fig. 4 B). In bars 2 and 4 in C, the expression levels of EGFP-PAG3 and ARF6-HA were essentially the same as those described in the legend to Fig. 2 and Fig. 3. Expression of ARF1-HA and ARF5-HA in A and B was also similar to those in Fig. 2. Data are expressed as the mean ± SE of three independent experiments. *P < 0.001.

Mentions: Plasmid DNA for PAG3 was cotransfected with plasmid DNA for ARF isoforms, where all plasmids were designed for the overexpression. As shown in Fig. 5 C, we found that increasing amounts of plasmid for EGFP-PAG3 together with decreasing amounts of plasmid for ARF6-HA gave bell-shaped dose responses of the phagocytosis index, and a condition was found where both EGFP-PAG3 and ARF6-HA were overexpressed but the phagocytic activity of the transfected cells was restored to almost normal. In contrast, a control experiment using the GAP-inactive mutant of EGFP-PAG3, instead of wild-type EGFP-PAG3, did not give such bell-shaped dose responses, but gave responses declining linearly in accordance with amounts of ARF6-HA plasmid DNA transfected (Fig. 5 C). Cooverexpression of EGFP-PAG3 with the Q67L or the T27N mutant of ARF6-HA was also unable to restore phagocytic activity (Fig. 5 C). Moreover, such conditions for the apparent restoration of the phagocytosis index were found neither with cooverexpression of EGFP-PAG3 and ARF1-HA nor with cooverexpression of EGFP-PAG3 and ARF5-HA (Fig. 5a and Fig. b), although we examined with several different ratios between PAG3 and these ARF isoforms in addition to those shown in Fig. 5 (data not shown). These results again are consistent with a notion that PAG3 acts as a GAP for the class III ARF, ARF6, but not other classes of ARFs, and is thereby involved in the FcγR-mediated phagocytosis in macrophages. Accumulation of F-actin beneath the IgG-opsonized beads was also observed in cells overexpressing both EGFP-PAG3 and ARF6-HA, where their phagocytic activities were restored to almost normal (Fig. 4 B).


PAG3/Papalpha/KIAA0400, a GTPase-activating protein for ADP-ribosylation factor (ARF), regulates ARF6 in Fcgamma receptor-mediated phagocytosis of macrophages.

Uchida H, Kondo A, Yoshimura Y, Mazaki Y, Sabe H - J. Exp. Med. (2001)

Cooverexpression of ARF6, but not other classes of ARF isoforms, restores the PAG3-induced inhibition of FcγR-mediated phagocytosis. P388D1 cells and cells transiently transfected with the indicated plasmid constructs in pEGFP and pcDNA3 vectors for overexpression were incubated with IgG-opsonized beads, and the phagocytosis index was determined, as described in the legend to Fig. 2. Plasmid DNAs for EGFP-PAG3 (WT) or its GAP-inactive mutant (CA) were cotransfected with plasmid DNAs for ARF1-HA (A), ARF5-HA (B), and ARF6-HA (C). In A–C, 2.0 μg of plasmid DNA for EGFP-PAG3 (bar 2), 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for each ARF-HA (bar 3), and 2.0 μg of plasmid DNA for each ARF-HA (bar 4) were used; the GAP-inactive mutant of EGFP-PAG3 was used instead of wild-type EGFP-PAG3 in bars 5–7, as indicated. In bars 8 and 9 in C, 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for the Q67L mutant (bar 8) or the T27N mutant (bar 9) of ARF6-HA were used. Mock-transfected controls were also included (bar 1 in A–C). The overexpression of exogenous proteins were confirmed as described in the legend to Fig. 2 and Fig. 3, with each cell examined for the phagocytic activities. In bar 3 in C, these cells expressed EGFP-PAG3 at levels 25–30-fold higher than endogenous PAG3, and expressed ARF6-HA at levels 7–9-fold higher than endogenous ARF6 (also see Fig. 4 B). In bars 2 and 4 in C, the expression levels of EGFP-PAG3 and ARF6-HA were essentially the same as those described in the legend to Fig. 2 and Fig. 3. Expression of ARF1-HA and ARF5-HA in A and B was also similar to those in Fig. 2. Data are expressed as the mean ± SE of three independent experiments. *P < 0.001.
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Figure 5: Cooverexpression of ARF6, but not other classes of ARF isoforms, restores the PAG3-induced inhibition of FcγR-mediated phagocytosis. P388D1 cells and cells transiently transfected with the indicated plasmid constructs in pEGFP and pcDNA3 vectors for overexpression were incubated with IgG-opsonized beads, and the phagocytosis index was determined, as described in the legend to Fig. 2. Plasmid DNAs for EGFP-PAG3 (WT) or its GAP-inactive mutant (CA) were cotransfected with plasmid DNAs for ARF1-HA (A), ARF5-HA (B), and ARF6-HA (C). In A–C, 2.0 μg of plasmid DNA for EGFP-PAG3 (bar 2), 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for each ARF-HA (bar 3), and 2.0 μg of plasmid DNA for each ARF-HA (bar 4) were used; the GAP-inactive mutant of EGFP-PAG3 was used instead of wild-type EGFP-PAG3 in bars 5–7, as indicated. In bars 8 and 9 in C, 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for the Q67L mutant (bar 8) or the T27N mutant (bar 9) of ARF6-HA were used. Mock-transfected controls were also included (bar 1 in A–C). The overexpression of exogenous proteins were confirmed as described in the legend to Fig. 2 and Fig. 3, with each cell examined for the phagocytic activities. In bar 3 in C, these cells expressed EGFP-PAG3 at levels 25–30-fold higher than endogenous PAG3, and expressed ARF6-HA at levels 7–9-fold higher than endogenous ARF6 (also see Fig. 4 B). In bars 2 and 4 in C, the expression levels of EGFP-PAG3 and ARF6-HA were essentially the same as those described in the legend to Fig. 2 and Fig. 3. Expression of ARF1-HA and ARF5-HA in A and B was also similar to those in Fig. 2. Data are expressed as the mean ± SE of three independent experiments. *P < 0.001.
Mentions: Plasmid DNA for PAG3 was cotransfected with plasmid DNA for ARF isoforms, where all plasmids were designed for the overexpression. As shown in Fig. 5 C, we found that increasing amounts of plasmid for EGFP-PAG3 together with decreasing amounts of plasmid for ARF6-HA gave bell-shaped dose responses of the phagocytosis index, and a condition was found where both EGFP-PAG3 and ARF6-HA were overexpressed but the phagocytic activity of the transfected cells was restored to almost normal. In contrast, a control experiment using the GAP-inactive mutant of EGFP-PAG3, instead of wild-type EGFP-PAG3, did not give such bell-shaped dose responses, but gave responses declining linearly in accordance with amounts of ARF6-HA plasmid DNA transfected (Fig. 5 C). Cooverexpression of EGFP-PAG3 with the Q67L or the T27N mutant of ARF6-HA was also unable to restore phagocytic activity (Fig. 5 C). Moreover, such conditions for the apparent restoration of the phagocytosis index were found neither with cooverexpression of EGFP-PAG3 and ARF1-HA nor with cooverexpression of EGFP-PAG3 and ARF5-HA (Fig. 5a and Fig. b), although we examined with several different ratios between PAG3 and these ARF isoforms in addition to those shown in Fig. 5 (data not shown). These results again are consistent with a notion that PAG3 acts as a GAP for the class III ARF, ARF6, but not other classes of ARFs, and is thereby involved in the FcγR-mediated phagocytosis in macrophages. Accumulation of F-actin beneath the IgG-opsonized beads was also observed in cells overexpressing both EGFP-PAG3 and ARF6-HA, where their phagocytic activities were restored to almost normal (Fig. 4 B).

Bottom Line: Overexpression of PAG3, but not its GAP-inactive mutant, attenuated the focal accumulation of F-actin and blocked phagocytosis, although surface levels of the FcgammaRs were not affected.Moreover, cooverexpression of ARF6, but not ARF1 or ARF5, restored the phagocytic activity of PAG3-overexpressing cells.We propose that PAG3 acts as a GAP for ARF6 and is hence involved in FcgammaR-mediated phagocytosis in mouse macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan.

ABSTRACT
The Fcgamma receptor (FcgammaR)-mediated phagocytosis of macrophages is a complex process where remodeling of both the actin-based cytoskeleton and plasma membrane occur coordinately. Several different families of small GTPases are involved. We have isolated a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF), paxillin-associated protein with ARFGAP activity (PAG)3/Papalpha/KIAA0400, from mature monocytes and macrophage-like cells. Mammalian ARFs fall into three classes, and the class III isoform (ARF6) has been shown to be involved in FcgammaR-mediated phagocytosis. Here we report that PAG3 is enriched together with ARF6 and F-actin at phagocytic cups formed beneath immunoglobulin G-opsonized beads in P388D1 macrophages, in which overexpression of ARF6, but not ARF1 (class I) or ARF5 (class II), inhibits the phagocytosis. Overexpression of PAG3, but not its GAP-inactive mutant, attenuated the focal accumulation of F-actin and blocked phagocytosis, although surface levels of the FcgammaRs were not affected. Other ubiquitously expressed ARFGAPs, G protein-coupled receptor kinase interactors GIT2 and GIT2-short/KIAA0148, which we have shown to exhibit GAP activity for ARF1 in COS-7 cells, did not accumulate at the phagocytic cups or inhibit phagocytosis. Moreover, cooverexpression of ARF6, but not ARF1 or ARF5, restored the phagocytic activity of PAG3-overexpressing cells. We propose that PAG3 acts as a GAP for ARF6 and is hence involved in FcgammaR-mediated phagocytosis in mouse macrophages.

Show MeSH
Related in: MedlinePlus