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Regulators and Effectors of Arf GTPases in Neutrophils.

Gamara J, Chouinard F, Davis L, Aoudjit F, Bourgoin SG - J Immunol Res (2015)

Bottom Line: In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses.GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells.The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Immunology, CHU de Quebec Research Center, Quebec, QC, Canada G1V 4G2.

ABSTRACT
Polymorphonuclear neutrophils (PMNs) are key innate immune cells that represent the first line of defence against infection. They are the first leukocytes to migrate from the blood to injured or infected sites. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of migrating PMNs. These phagocytes actively engulf microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens with bactericidal compounds. Association of the NADPH oxidase complex at the phagosomal membrane for production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. G protein-dependent signalling pathways tightly control PMN functions. In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses. GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells. The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology.

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Related in: MedlinePlus

AGAP2 efficiently stimulates GTP hydrolysis on Arf1 and GAP activity is stimulated by products of PI3K, PtdIns(3)P, and PtdIns(3,5)P2. Recombinant myristoylated Arf1 was purified from E. coli as previously described [20]. AGAP2 cDNA was inserted into the pACHLT-A baculovirus shuttle vector and cotransfected with linearized BaculoGold viral DNA into sf9 cells. Culture supernatants were used to infect sf9 cells with an MOI of 10. Insect cells were collected 48 h after infection and His6-AGAP2 was purified from sf9 lysates by chromatography on Ni-trap columns. (a) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (molar ratio 40.55 : 31 : 28.45) for 30 min at 30°C. AGAP2 at the indicated concentrations was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated for 30 min at 30°C in GAP buffer (20 mM Tris pH 8.0, 2 mM DTT, 100 mM NaCl, 1 mM MgCl2, and 100 μg/mL liposomes). (b) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (molar ratio 40.55 : 31 : 28.45), and liposome-supplemented PtdIns(3)P or PtdIns(3,5)P2 (molar ratio 37.4 : 28.5 : 26.2 : 7.9) for 30 min at 30°C. AGAP2 (10 nM) was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated at 30°C in GAP buffer for indicated time points. Reactions were stopped by dilution in ice-cold stop buffer (20 mM Tris pH 8.0, 1 mM DTT, and 10 mM MgCl2). Samples were filtered on Gelman GN-6 membranes and bound nucleotides were eluted with 2 M LiCl. GTP was separated from GDP by chromatography using polyethylenimine cellulose TLC plates developed in 1 M LiCl/1 M formic acid. The GTPα32P/GDPα32P ratios were calculated after exposure of TLC plates to a phosphorimager.
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Related In: Results  -  Collection


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fig4: AGAP2 efficiently stimulates GTP hydrolysis on Arf1 and GAP activity is stimulated by products of PI3K, PtdIns(3)P, and PtdIns(3,5)P2. Recombinant myristoylated Arf1 was purified from E. coli as previously described [20]. AGAP2 cDNA was inserted into the pACHLT-A baculovirus shuttle vector and cotransfected with linearized BaculoGold viral DNA into sf9 cells. Culture supernatants were used to infect sf9 cells with an MOI of 10. Insect cells were collected 48 h after infection and His6-AGAP2 was purified from sf9 lysates by chromatography on Ni-trap columns. (a) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (molar ratio 40.55 : 31 : 28.45) for 30 min at 30°C. AGAP2 at the indicated concentrations was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated for 30 min at 30°C in GAP buffer (20 mM Tris pH 8.0, 2 mM DTT, 100 mM NaCl, 1 mM MgCl2, and 100 μg/mL liposomes). (b) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (molar ratio 40.55 : 31 : 28.45), and liposome-supplemented PtdIns(3)P or PtdIns(3,5)P2 (molar ratio 37.4 : 28.5 : 26.2 : 7.9) for 30 min at 30°C. AGAP2 (10 nM) was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated at 30°C in GAP buffer for indicated time points. Reactions were stopped by dilution in ice-cold stop buffer (20 mM Tris pH 8.0, 1 mM DTT, and 10 mM MgCl2). Samples were filtered on Gelman GN-6 membranes and bound nucleotides were eluted with 2 M LiCl. GTP was separated from GDP by chromatography using polyethylenimine cellulose TLC plates developed in 1 M LiCl/1 M formic acid. The GTPα32P/GDPα32P ratios were calculated after exposure of TLC plates to a phosphorimager.

Mentions: In humans, 11 genes are predicted to encode for AGAP-type Arf GAPs [127], with AGAP1 and AGAP2 being the most studied. AGAP1/2 have high GAP activity toward Arf1 and Arf5 and weak activity towards Arf6 [165, 166]. GAP activity is stimulated by PtdIns(4,5)P2 and phosphatidic acid as well [165, 166]. The AGAP2 gene encodes for three protein isoforms; PIKE-L and PIKE-S, which are restricted to brain, whereas PIKE-A (AGAP2) is more ubiquitously expressed [167, 168]. As shown in Figure 4(a), purified recombinant AGAP2 is a very potent Arf1 GAP. GAP activity is strongly stimulated by PtdIns(3)P and PtdIns(3,5)P2, the products of PI3Ks (Figure 4(b)). AGAP2 was reported to colocalize with AP-1 and transferrin receptors on recycling endosomes, and, together with Arf1, to regulate retrograde trafficking between early endosomes and the TGN [166, 169]. Moreover, AGAP2 plays a role in the signalling pathways and regulates the recycling of β2-adrenergic receptors [170]. During cell migration, AGAP2 was shown to promote focal adhesion disassembly through binding to and stimulation of focal adhesion kinase [171]. We generated polyclonal AGAP2 antibodies that detect a protein of about 90 kDa in PMNs (Figure 5(a)). The 90 kDa protein recovered in 1% NP-40 PMN lysates was immunoprecipitated by the AGAP2 antibody but not by the preimmune serum (Figure 5(b)). The band was analysed by mass spectrometry. Overall, 32 peptides covering 44% of the AGAP2 amino acid sequence were identified. Among these peptides, two were unique to AGAP2 and there were no signature peptides for AGAP1 or PIKE-L (Figure 5(c)). Taken together, the data indicate that AGAP2, but not AGAP1 or PIKE-L, was expressed in PMNs. This work is still in progress, but preliminary observations suggest that AGAP2 regulates phagocytosis independently of its GAP activity.


Regulators and Effectors of Arf GTPases in Neutrophils.

Gamara J, Chouinard F, Davis L, Aoudjit F, Bourgoin SG - J Immunol Res (2015)

AGAP2 efficiently stimulates GTP hydrolysis on Arf1 and GAP activity is stimulated by products of PI3K, PtdIns(3)P, and PtdIns(3,5)P2. Recombinant myristoylated Arf1 was purified from E. coli as previously described [20]. AGAP2 cDNA was inserted into the pACHLT-A baculovirus shuttle vector and cotransfected with linearized BaculoGold viral DNA into sf9 cells. Culture supernatants were used to infect sf9 cells with an MOI of 10. Insect cells were collected 48 h after infection and His6-AGAP2 was purified from sf9 lysates by chromatography on Ni-trap columns. (a) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (molar ratio 40.55 : 31 : 28.45) for 30 min at 30°C. AGAP2 at the indicated concentrations was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated for 30 min at 30°C in GAP buffer (20 mM Tris pH 8.0, 2 mM DTT, 100 mM NaCl, 1 mM MgCl2, and 100 μg/mL liposomes). (b) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (molar ratio 40.55 : 31 : 28.45), and liposome-supplemented PtdIns(3)P or PtdIns(3,5)P2 (molar ratio 37.4 : 28.5 : 26.2 : 7.9) for 30 min at 30°C. AGAP2 (10 nM) was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated at 30°C in GAP buffer for indicated time points. Reactions were stopped by dilution in ice-cold stop buffer (20 mM Tris pH 8.0, 1 mM DTT, and 10 mM MgCl2). Samples were filtered on Gelman GN-6 membranes and bound nucleotides were eluted with 2 M LiCl. GTP was separated from GDP by chromatography using polyethylenimine cellulose TLC plates developed in 1 M LiCl/1 M formic acid. The GTPα32P/GDPα32P ratios were calculated after exposure of TLC plates to a phosphorimager.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4644846&req=5

fig4: AGAP2 efficiently stimulates GTP hydrolysis on Arf1 and GAP activity is stimulated by products of PI3K, PtdIns(3)P, and PtdIns(3,5)P2. Recombinant myristoylated Arf1 was purified from E. coli as previously described [20]. AGAP2 cDNA was inserted into the pACHLT-A baculovirus shuttle vector and cotransfected with linearized BaculoGold viral DNA into sf9 cells. Culture supernatants were used to infect sf9 cells with an MOI of 10. Insect cells were collected 48 h after infection and His6-AGAP2 was purified from sf9 lysates by chromatography on Ni-trap columns. (a) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (molar ratio 40.55 : 31 : 28.45) for 30 min at 30°C. AGAP2 at the indicated concentrations was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated for 30 min at 30°C in GAP buffer (20 mM Tris pH 8.0, 2 mM DTT, 100 mM NaCl, 1 mM MgCl2, and 100 μg/mL liposomes). (b) GTPα32P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (molar ratio 40.55 : 31 : 28.45), and liposome-supplemented PtdIns(3)P or PtdIns(3,5)P2 (molar ratio 37.4 : 28.5 : 26.2 : 7.9) for 30 min at 30°C. AGAP2 (10 nM) was mixed with 0.3 μM GTPα32P-loaded Arf1 and incubated at 30°C in GAP buffer for indicated time points. Reactions were stopped by dilution in ice-cold stop buffer (20 mM Tris pH 8.0, 1 mM DTT, and 10 mM MgCl2). Samples were filtered on Gelman GN-6 membranes and bound nucleotides were eluted with 2 M LiCl. GTP was separated from GDP by chromatography using polyethylenimine cellulose TLC plates developed in 1 M LiCl/1 M formic acid. The GTPα32P/GDPα32P ratios were calculated after exposure of TLC plates to a phosphorimager.
Mentions: In humans, 11 genes are predicted to encode for AGAP-type Arf GAPs [127], with AGAP1 and AGAP2 being the most studied. AGAP1/2 have high GAP activity toward Arf1 and Arf5 and weak activity towards Arf6 [165, 166]. GAP activity is stimulated by PtdIns(4,5)P2 and phosphatidic acid as well [165, 166]. The AGAP2 gene encodes for three protein isoforms; PIKE-L and PIKE-S, which are restricted to brain, whereas PIKE-A (AGAP2) is more ubiquitously expressed [167, 168]. As shown in Figure 4(a), purified recombinant AGAP2 is a very potent Arf1 GAP. GAP activity is strongly stimulated by PtdIns(3)P and PtdIns(3,5)P2, the products of PI3Ks (Figure 4(b)). AGAP2 was reported to colocalize with AP-1 and transferrin receptors on recycling endosomes, and, together with Arf1, to regulate retrograde trafficking between early endosomes and the TGN [166, 169]. Moreover, AGAP2 plays a role in the signalling pathways and regulates the recycling of β2-adrenergic receptors [170]. During cell migration, AGAP2 was shown to promote focal adhesion disassembly through binding to and stimulation of focal adhesion kinase [171]. We generated polyclonal AGAP2 antibodies that detect a protein of about 90 kDa in PMNs (Figure 5(a)). The 90 kDa protein recovered in 1% NP-40 PMN lysates was immunoprecipitated by the AGAP2 antibody but not by the preimmune serum (Figure 5(b)). The band was analysed by mass spectrometry. Overall, 32 peptides covering 44% of the AGAP2 amino acid sequence were identified. Among these peptides, two were unique to AGAP2 and there were no signature peptides for AGAP1 or PIKE-L (Figure 5(c)). Taken together, the data indicate that AGAP2, but not AGAP1 or PIKE-L, was expressed in PMNs. This work is still in progress, but preliminary observations suggest that AGAP2 regulates phagocytosis independently of its GAP activity.

Bottom Line: In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses.GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells.The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Immunology, CHU de Quebec Research Center, Quebec, QC, Canada G1V 4G2.

ABSTRACT
Polymorphonuclear neutrophils (PMNs) are key innate immune cells that represent the first line of defence against infection. They are the first leukocytes to migrate from the blood to injured or infected sites. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of migrating PMNs. These phagocytes actively engulf microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens with bactericidal compounds. Association of the NADPH oxidase complex at the phagosomal membrane for production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. G protein-dependent signalling pathways tightly control PMN functions. In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses. GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells. The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology.

Show MeSH
Related in: MedlinePlus