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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

Expression of GIT2 in immune cells. RAW264 macrophages (1.5 × 107 cells), human PMNs (3 × 107), human lymphocytes/monocytes (LM, 3 × 107), and dimethyl sulfoxide-differentiated HL-60 cells (3 × 107) were mixed with an equal volume of boiling denaturing buffer and cell lysates were processed essentially as described by Marcil et al. [19]. The supernatants were then filtered through Sephadex G-10 columns to remove the denaturing agents and 0.1% Nonidet P-40, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 5 μL of bovine serum albumin (0.01% w/v) were added to the eluates. Samples were precleared with protein A-Sepharose and subsequently used for overnight immunoprecipitation with the polyclonal GIT2 antibodies 10 and 11 (5 μL). The beads were washed three times with ice-cold nondenaturing lysis buffer containing 1% Nonidet P-40 and boiled for 7 min at 100°C in 2x Laemmli's sample buffer as described previously [19]. Immunoprecipitated proteins were electrophoresed on 10% SDS-PAGE and proteins were transferred to Immobilon PVDF membrane (Millipore Corp., Bedford, MA, USA). Membranes were incubated with the p95PKL/GIT2 antibody (P94020; 1 : 1500) from Bection Dickinson (Mississauga, ON, Canada) and exposed to peroxidase-conjugated anti-rabbit IgG (1 : 20,000) for 1 h at 37°C. The membranes were covered with ECL+ detection reagents. Images were obtained by exposing Kodak X-Omat film to membranes for 20 sec (upper panel) and 5 min (lower panel).
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fig2: Expression of GIT2 in immune cells. RAW264 macrophages (1.5 × 107 cells), human PMNs (3 × 107), human lymphocytes/monocytes (LM, 3 × 107), and dimethyl sulfoxide-differentiated HL-60 cells (3 × 107) were mixed with an equal volume of boiling denaturing buffer and cell lysates were processed essentially as described by Marcil et al. [19]. The supernatants were then filtered through Sephadex G-10 columns to remove the denaturing agents and 0.1% Nonidet P-40, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 5 μL of bovine serum albumin (0.01% w/v) were added to the eluates. Samples were precleared with protein A-Sepharose and subsequently used for overnight immunoprecipitation with the polyclonal GIT2 antibodies 10 and 11 (5 μL). The beads were washed three times with ice-cold nondenaturing lysis buffer containing 1% Nonidet P-40 and boiled for 7 min at 100°C in 2x Laemmli's sample buffer as described previously [19]. Immunoprecipitated proteins were electrophoresed on 10% SDS-PAGE and proteins were transferred to Immobilon PVDF membrane (Millipore Corp., Bedford, MA, USA). Membranes were incubated with the p95PKL/GIT2 antibody (P94020; 1 : 1500) from Bection Dickinson (Mississauga, ON, Canada) and exposed to peroxidase-conjugated anti-rabbit IgG (1 : 20,000) for 1 h at 37°C. The membranes were covered with ECL+ detection reagents. Images were obtained by exposing Kodak X-Omat film to membranes for 20 sec (upper panel) and 5 min (lower panel).

Mentions: The GIT subfamily includes the two structurally related proteins GIT1 and GIT2 [127]. They possess the zinc-finger motif required for their GAP activity on Arf6 [144]. Though GIT proteins have no PH domain, their GAP activity was reported to be stimulated by PtdIns(3,4,5)P3 [144]. GIT1 interacts with various GPCRs to regulate their endocytosis via the clathrin pathway in a G protein-coupled receptor kinase, β-arrestin, and dynamin-dependent manner [145, 146]. It is worth highlighting that GIT proteins can form complexes with PIX, a GEF specific for Rho GTPases Rac2 and Cdc42 [147]. GIT/PIX complexes regulate Cdc42/Rac-dependent activation of p21-activated kinase 1 (PAK1), a protein involved in microtubule-mediated focal adhesion disassembly [148]. GIT2 and a splice variant named GIT2-short have been characterized, with the expression of the latter being restricted to immune cells [149]. Overexpression of GIT2-short was reported to cause redistribution of Golgi protein β-COP, to affect the subcellular localization of paxillin, and to reduce the levels of actin-based fibers [150]. GIT2 is expressed in human lymphocytes and/or monocytes, mature PMNs, HL-60 promyelocytic leukemia cells, and the rat macrophage cell line RAW264 (Figure 2). In PMNs obtained from GIT2-deficient mice, Arf1 was reported to be hyperactivated in response to stimulation with fMLF [45]. GIT2 deficiency was associated with reduced directional migration to fMLF and enhanced production of superoxide even if NADPH oxidase polarization at the leading edge of migrating PMNs was lost [45]. Interestingly, the Arf GEF GBF1 has been suggested to control the activation of Arf1 and to target p22phox and GIT2 to the leading edge of chemotaxing PMNs [44].


Regulators and Effectors of Arf GTPases in Neutrophils.

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

Expression of GIT2 in immune cells. RAW264 macrophages (1.5 × 107 cells), human PMNs (3 × 107), human lymphocytes/monocytes (LM, 3 × 107), and dimethyl sulfoxide-differentiated HL-60 cells (3 × 107) were mixed with an equal volume of boiling denaturing buffer and cell lysates were processed essentially as described by Marcil et al. [19]. The supernatants were then filtered through Sephadex G-10 columns to remove the denaturing agents and 0.1% Nonidet P-40, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 5 μL of bovine serum albumin (0.01% w/v) were added to the eluates. Samples were precleared with protein A-Sepharose and subsequently used for overnight immunoprecipitation with the polyclonal GIT2 antibodies 10 and 11 (5 μL). The beads were washed three times with ice-cold nondenaturing lysis buffer containing 1% Nonidet P-40 and boiled for 7 min at 100°C in 2x Laemmli's sample buffer as described previously [19]. Immunoprecipitated proteins were electrophoresed on 10% SDS-PAGE and proteins were transferred to Immobilon PVDF membrane (Millipore Corp., Bedford, MA, USA). Membranes were incubated with the p95PKL/GIT2 antibody (P94020; 1 : 1500) from Bection Dickinson (Mississauga, ON, Canada) and exposed to peroxidase-conjugated anti-rabbit IgG (1 : 20,000) for 1 h at 37°C. The membranes were covered with ECL+ detection reagents. Images were obtained by exposing Kodak X-Omat film to membranes for 20 sec (upper panel) and 5 min (lower panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Expression of GIT2 in immune cells. RAW264 macrophages (1.5 × 107 cells), human PMNs (3 × 107), human lymphocytes/monocytes (LM, 3 × 107), and dimethyl sulfoxide-differentiated HL-60 cells (3 × 107) were mixed with an equal volume of boiling denaturing buffer and cell lysates were processed essentially as described by Marcil et al. [19]. The supernatants were then filtered through Sephadex G-10 columns to remove the denaturing agents and 0.1% Nonidet P-40, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 5 μL of bovine serum albumin (0.01% w/v) were added to the eluates. Samples were precleared with protein A-Sepharose and subsequently used for overnight immunoprecipitation with the polyclonal GIT2 antibodies 10 and 11 (5 μL). The beads were washed three times with ice-cold nondenaturing lysis buffer containing 1% Nonidet P-40 and boiled for 7 min at 100°C in 2x Laemmli's sample buffer as described previously [19]. Immunoprecipitated proteins were electrophoresed on 10% SDS-PAGE and proteins were transferred to Immobilon PVDF membrane (Millipore Corp., Bedford, MA, USA). Membranes were incubated with the p95PKL/GIT2 antibody (P94020; 1 : 1500) from Bection Dickinson (Mississauga, ON, Canada) and exposed to peroxidase-conjugated anti-rabbit IgG (1 : 20,000) for 1 h at 37°C. The membranes were covered with ECL+ detection reagents. Images were obtained by exposing Kodak X-Omat film to membranes for 20 sec (upper panel) and 5 min (lower panel).
Mentions: The GIT subfamily includes the two structurally related proteins GIT1 and GIT2 [127]. They possess the zinc-finger motif required for their GAP activity on Arf6 [144]. Though GIT proteins have no PH domain, their GAP activity was reported to be stimulated by PtdIns(3,4,5)P3 [144]. GIT1 interacts with various GPCRs to regulate their endocytosis via the clathrin pathway in a G protein-coupled receptor kinase, β-arrestin, and dynamin-dependent manner [145, 146]. It is worth highlighting that GIT proteins can form complexes with PIX, a GEF specific for Rho GTPases Rac2 and Cdc42 [147]. GIT/PIX complexes regulate Cdc42/Rac-dependent activation of p21-activated kinase 1 (PAK1), a protein involved in microtubule-mediated focal adhesion disassembly [148]. GIT2 and a splice variant named GIT2-short have been characterized, with the expression of the latter being restricted to immune cells [149]. Overexpression of GIT2-short was reported to cause redistribution of Golgi protein β-COP, to affect the subcellular localization of paxillin, and to reduce the levels of actin-based fibers [150]. GIT2 is expressed in human lymphocytes and/or monocytes, mature PMNs, HL-60 promyelocytic leukemia cells, and the rat macrophage cell line RAW264 (Figure 2). In PMNs obtained from GIT2-deficient mice, Arf1 was reported to be hyperactivated in response to stimulation with fMLF [45]. GIT2 deficiency was associated with reduced directional migration to fMLF and enhanced production of superoxide even if NADPH oxidase polarization at the leading edge of migrating PMNs was lost [45]. Interestingly, the Arf GEF GBF1 has been suggested to control the activation of Arf1 and to target p22phox and GIT2 to the leading edge of chemotaxing PMNs [44].

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