rapid recruitment of innate immunity cells such as polymorphonuclear neutrophils ( pmns ) is a critical component of pathogen killing and removal during infection .
as they differentiate , the cells begin to move toward the venous sinusoids prior to migration across the sinusoidal endothelium to reach the vascular lumen .
these terminally differentiated cells have a short life   but nevertheless represent the most abundant leukocyte species in the circulation .
pmns are the first leukocytes to migrate from the blood to inflammatory sites [ 2 , 3 ] .
following their activation by various proinflammatory cytokines such as il-8 , tnf , or il-1 secreted by resident macrophages , pmns start rolling along the vessel wall , followed by firm arrest and transmigration through the inflammatory vascular endothelium [ 46 ] .
once in the extravascular environment , pmns interact with extracellular matrix proteins and migrate along a chemotactic gradient to reach the site of injury .
at the site of infection , pmns begin phagocytosis and killing of pathogens through production of toxic reactive oxygen species ( ros ) , secretion of lysosomal enzymes , and formation of nets ( figure 1(a ) ) [ 2 , 8 ] . activated pmns
also regulate the innate and the adaptive immune responses by secretion of various cytokines and chemokines , such as il-1 , il-6 , il-8 , tnf [ 2 , 911 ] , and lipid mediators as well .
the interaction of pmns with their environment is an indispensable determinant that tailors their functional responses , including correct timing of events leading to activation .
the mechanisms that contribute to the maintenance of pmn homeostasis under normal and inflammatory conditions are tightly regulated through integration of external signals picked up by their transmembrane receptors .
these receptors mediate intracellular signalling cascades through activation of two superfamilies of g proteins , the heterotrimeric g proteins , and the ras superfamily of small monomeric gtpases [ 13 , 14 ] .
whereas heterotrimeric g proteins directly interact with and are activated following stimulation of so - called g protein - coupled receptors ( gpcrs ) such as formyl - peptide receptors or cxcr1 chemokine receptor , small gtpases are generally activated by other regulatory proteins downstream of many transmembrane receptors ( figure 1(b ) ) .
the ras superfamily comprises more than 150 members divided into six subfamilies : ras , rho , ran , rab , rheb , and arf .
small gtpases are molecular switches that exist in an active  on  form when bound to guanosine - triphosphate ( gtp ) and an inactive  off  conformation when bound to guanosine - diphosphate ( gdp ) [ 15 , 16 ] .
the guanine nucleotide exchange factors ( gefs ) catalyze the removal of gdp and allow gtp binding to the conserved guanine nucleotide binding site of small gtpases .
the binding of gtp rigidifies small gtpases in an active conformation that interacts with specific effector proteins and engages a limited number of downstream effects .
small gtpases have low intrinsic gtpase activity and need help from gtpase activating proteins ( gaps ) to hydrolyse gtp and return to the inactive gdp - bound conformation [ 17 , 18 ] .
some gtpases of the rho and rab family are also regulated by guanine nucleotide dissociation inhibitors ( gdis ) that remove small gtpases from the membranes and sequester them in the inactive state as a cytosolic heterodimer .
small gtpases are key elements of downstream signalling pathways regulating multiple effector proteins and functional responses of cells . numerous studies , including those from our laboratory , have shown that arf proteins activate phospholipase d ( pld ) and phosphatidylinositol 4-phosphate 5-kinase ( pip5-kinase ) and regulate various pmn functions such as superoxide production , degranulation , and chemotaxis ( figure 1(b ) and table 1 ) . in this review , we will focus on the arf gtpases and their regulators in relation to pmn functional responses .
the arf family was first identified and named according to its function as a cholera toxin cofactor stimulating the adp - ribosylation of g protein g subunit .
arf gtpases are ubiquitously expressed and their sequences are highly conserved among eukaryotes . they are divided into three classes based on sequence homology .
class ii comprises arf4 and arf5 , and arf6 is the only representative of class iii [ 23 , 24 ] .
we can not overlook the fact that small g proteins sharing structural features with arfs also include arf - like ( arl ) proteins , ras - related protein 1 ( sar1 ) , and arf - related protein 1 ( arfrp1 ) . like all small gtpases , arfs are on - off molecular switches regulated by specific gaps and gefs .
in contrast to other small g proteins that undergo posttranslational modification ( prenylation or isoprenylation ) at their c - terminus , arfs share n - terminal amphipathic helix with a myristoylated n - terminal glycine residue .
gtp binding to arfs induces conformational changes in switch 1 and switch 2 regions that bind effector proteins , as well as a reorientation of the amphipathic helix that favours interaction with the membrane and insertion of the lipid tail into the phospholipid bilayer .
arfs also have different distribution in the cells , which is thought to stem from the individual protein environment .
although classes i and ii arfs are mainly localized to the er - golgi , arf1 and arf3 are released in the cytosol , whereas arf4 and arf5 can associate with the golgi and the trans - golgi network ( tgn ) in their gdp - bound state .
studies have determined that the gdp - gtp cycle of arf6 takes place at the plasma membrane and that some gtp - binding defective mutants of arf6 are trapped in endosomes [ 30 , 31 ] .
the two that have been the most studied are arf1 and arf6 ( table 1 ) .
arf1 was first reported to regulate intracellular vesicular traffic from the golgi to the endoplasmic reticulum ( er ) and between golgi cisternae , through the recruitment of clathrin and nonclathrin coats to membrane , a first step in the budding of transport vesicles [ 5052 ] .
in addition to stimulating pld , golgi - associated arfs recruit phosphatidylinositol 4-kinase and pip5-kinase to maintain the structure and the dynamics of the golgi apparatus through local synthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate ( ptdins(4,5)p2 ) .
arf1 and arf3 are required for the integrity of recycling endosomes but seem dispensable for the retrograde transport from endosomal compartments to the tgn .
, arf1 was shown to mediate formyl - methionyl - leucyl - phenylalanine ( fmlf ) dependent activation of pld .
studies from our laboratory have shown that the particulate agonist monosodium urate crystals , fmlf , and leukotriene b4 induce arf1 recruitment to membranes and pld activation in pmns .
a study using cytosol - depleted hl-60 cells suggested that arf1 and phosphatidylinositol transfer protein ( pitp ) restore secretory function in cytosol - depleted cells by promoting ptdins(4,5)p2 synthesis .
ptdins(4,5)p2 synthesis could also be dependent on arf1-mediated activation of pip5-kinase in hl-60 cells . in pmns
, arf1 was shown to bind to complement receptor type 1 ( cr1 ) storage vesicles and was suggested to play a role in regulation of their transport .
though activated arf1 has been shown to recruit arfaptin-1 and arfaptin-2 to golgi membranes and has been suggested to regulate golgi function in hl-60 cells , more recent studies showed that arl1 but not other arf proteins determine the association of arfaptins with and the biogenesis of secretory granules at the trans - golgi in cells [ 59 , 60 ] .
less is known about the function of arf class ii . arf4 and arf5 play roles in transport mechanisms between the endoplasmic reticulum golgi intermediate compartment ( ergic ) and the golgi .
arf4 was reported to regulate transport of ciliary cargoes , and the creb3-arf4 signalling cascade was suggested to be part of a golgi stress response to pathogens . in hela cells , arf5 was reported to regulate internalization of the 51 integrin and clathrin - mediated endocytosis of specific cargoes . though quantitative proteomics studies have identified arf4 and arf5 in pmns [ 47 , 65 ] , the functions of arf class ii in pmns have not been investigated .
arf6 , the arf gtpase most distantly related to arf1 , has been localized to the plasma membrane and endosomal compartments .
this gtpase has been implicated in different signalling pathways and in a wide diversity of cellular functions such as actin cytoskeleton remodelling , phagocytosis , endocytosis , membrane receptor recycling , and intracellular transport [ 52 , 6670 ] .
overexpression of arf6 and of its regulators in metastatic cancers suggests important roles in regulating adhesion , migration , and invasive behaviour of cancer cells [ 7173 ] .
for example , in epithelial cells , e - cadherin is targeted by arf6 to adherens junctions for maintaining barrier permeability , epithelial cell morphology , and polarity [ 74 , 75 ] . in immune cells such as macrophages ,
a spatiotemporal recruitment of arf6 to phagosomes regulates fc receptor - dependent phagocytosis [ 7679 ] .
this gtpase is also involved in endocytosis and recycling of various gpcrs [ 8082 ] such as mu - opioid   and 2-adrenergic   receptors , or growth factor receptors as well .
moreover , it is important to mention that arf6 plays a major role in the signalling pathway of toll - like receptor 4 ( tlr4 ) and tlr9 [ 86 , 87 ] .
the expression of arf6 in pmns and neutrophil - like cells such as differentiated hl-60 or plb-985 myeloid leukemia cells has been previously reported [ 3638 ] .
arf6 protein is four to five times more abundant in these myeloid leukemia cells when compared to human pmns . in pmns or differentiated plb-985 cells
, arf6 plays roles in the signalling pathways elicited by the chemotactic peptide fmlf [ 37 , 38 ] .
plb-985 cells , overexpressing the arf6 ( q67l ) mutant defective in gtp hydrolysis or the arf6 ( t27n ) mutant defective in gtp binding , show increased and decreased nadph oxidase activity , respectively .
silencing of arf6 in plb-985 cells also reduces fmlf - mediated production of superoxide and pld activation as well .
inhibition of superoxide production by arf6 mutants could be due to reduced pld activity since overexpression of the arf6 ( n48r ) mutant defective in pld activation also reduced fmlf - induced nadph oxidase activity in plb-985 cells .
in addition to pld , pip5-kinase was reported to be a downstream effector of arf6 [ 88 , 89 ] . in this context
, it is important to highlight that arf proteins can be depleted from hl-60 cells by permeabilization and that addition of arf6 to permeabilized cells contributes to the regulation of pdtins(4,5)p2 synthesis at the plasma membrane by directly activating pip5-kinase .
altogether , these studies suggest that remodelling of membrane phospholipids by arf6-mediated activation of pld enzymes and pip5-kinase would regulate pmn functional responses such as nadph oxidase activity , phagocytosis , and degranulation [ 9092 ] . direct demonstration of a role for arf6 in pmn phagocytosis and degranulation awaits characterization of arf6 knockout pmns .
gefs can signal from plasma membrane receptors directly to small gtpases , and in some cases gefs serve as gtpase effectors or adaptor proteins that facilitate activation of other small gtpase family members .
the human genome contains gefs that are family specific but some gefs are highly specific toward one gtpase .
the first characterized arf gefs comprise the yeast gea1p and in mammals cytohesin-2 and big1 [ 9496 ] .
although arf gefs show different substrate specificities , they share a conserved 200-amino acid region called the sec7 domain that catalyzes the exchange of nucleotides . some but not all sec7 domains are the target of the drug brefeldin a ( bfa ) .
arf gefs contain other motifs , like the pleckstrin homology ( ph ) domain involved in protein targeting to membranes through binding to polyphosphoinositides , homology downstream of sec7 ( hds ) lipid - binding domains , or sh2 and sh3 domains related to protein - protein interactions .
arf gefs are classified into six evolutionarily conserved families as follows : gbf1 , big , psd , iqsec , cytohesins , and fbxo8 . the golgi - specific bfa resistance factor 1 ( gbf1 ) and the two yeast gbf1 orthologs , gea1 and gea2 , localize in the golgi .
lipid binding to the hds1 domain immediately downstream of the sec7 domain is sufficient for targeting gbf1 to lipid droplets and golgi membranes .
gbf1 recruits the copi coat to the cis - golgi [ 99101 ] . in vitro
gbf1 has been reported to activate arf1 in response to stimulation with fmlf . upon stimulation , arf1 and gbf1
are relocalized from the golgi to the leading edge of migrating cells in a phosphatidylinositol 3-kinase- ( pi3k- ) dependent manner .
the silencing of gbf1 in hl-60 cells abrogates cell polarisation , direction sensing , and superoxide production induced by fmlf .
the big ( bfa inhibited gef ) family of arf gefs comprises big1 and big2 in mammals .
big2 has been shown to activate class i arfs ( arf1 and arf3 ) and to localize to the tgn and recycling endosomes [ 103106 ] .
big1 and big2 have redundant functions and are important determinants of arf - based membrane traffic between the tgn and late endosomes .
in addition to lipid binding , the hds1 domain of sec7 , the yeast orthologue of big2/big2 , is important to localize this arf gef to golgi membrane compartments on which arf1 has already been activated . a cascade in which gbf1-activated arf4 and arf5 regulate the recruitment of big1 and big2 to the tgn has been reported .
this study provides a mechanistic basis for the effects of a combination of arf class i and class ii knockdowns on golgi morphology .
although bfa has been reported to inhibit fmlf - mediated production of superoxide in pmns , the bfa sensitive arf gef involved in this effect , if any , has not been characterized .
the psd family of arf gefs , also known as efa6 ( exchange factor for arf6 ) , comprises four paralogs in vertebrates ( efa6a , efa6b , efa6c , and efa6d ) .
efa6 is plasma membrane - targeted through interaction with ptdins(4,5)p2 and f - actin [ 111 , 112 ] .
efa6 is involved in cytoskeletal rearrangement and clathrin - mediated endocytosis [ 112 , 113 ] .
the bfa resistant arf gefs ( brags ) or iqsec family contains three members in vertebrates .
brag1/2 were found associated to endosomes and were found to localize with arf6 at the cell periphery [ 114116 ] .
in addition to arf6 , brag2 was reported to activate arf4 and arf5 and to regulate arf5-dependent internalization of 1 integrins in the clathrin - coated pits .
other studies have shown that brag2 plays a role in cell adhesion and phagocytosis through regulation of  integrin trafficking in epithelial cells and monocytes , respectively [ 117 , 118 ] .
other arf family gefs include sec12 , a type ii er membrane protein that is a specific sar1 gef , and the f - box protein 8 gene family ( fbxo8 ) .
fbxo8 might not function as a gef but as a factor that controls the intracellular levels of arf6 protein through ubiquitinylation - mediated proteasomal degradation .
the cytohesin ( cyth ) family is represented by four bfa - insensitive arf gefs in vertebrates .
the structural organization of cyths includes n - terminal coiled - coil domain involved in cyth dimerization or protein - protein interaction , the sec7 domain , and a c - terminal ph domain [ 122 , 123 ] .
the affinity of their ph domain for ptdins(4,5)p2 and phosphatidylinositol 3,4,5-trisphosphate ( ptdins(3,4,5)p3 ) is an important determinant for cyth localization and/or recruitment to the plasma membrane .
studies from our laboratory have reported the expression of cytohesin-2/arno ( cyth-2 ) and cytohesin-3 ( cyth-3 ) in undifferentiated hl-60 or plb-985 cells and a strong induction of cytohesin-1 ( cyth-1 ) expression during granulocytic differentiation [ 20 , 39 ] . in differentiated hl-60 cells ,
stimulation with fmlf induced a pi3k - independent and pi3k - dependent membrane recruitment of cyth-1 and cyth-2 , respectively .
we have previously shown that pharmacological inhibition of cyth-1 with secinh3 inhibited fmlf - mediated membrane translocation of arf6 and arf1 and activation of arf6 in pmns .
studies using secinh3 in pmns and plb-985 cells overexpressing cyth-1 or silenced for arf6 have highlighted a role for the cyth-1-arf6 signalling axis in pld activation and two major bactericidal functions , degranulation and nadph oxidase activity .
furthermore , pharmacological inhibition or silencing of cyth-1 was shown to reduce the internalization of fprl-1 ( formyl - peptide - like receptor 1 ) in fmlf activated granulocytes .
cyth-1 was initially characterized as a positive regulator of the 2 integrin lfa-1 ( lymphocyte function antigen-1 ) functions in lymphocytes [ 4042 ] . in our laboratory
, we showed that overexpression of cyth-1 in plb-985 cells increases lfa-1-dependent adhesion to endothelial cells .
in contrast , plb-985 cells silenced for cyth-1 and pmns treated with secinh3 show decreased lfa-1-dependent adhesion to endothelial cells .
further studies from our laboratory also documented that cyth-1 associates with and restrains the activation of the 2 integrin mac-1 ( macrophage antigen-1 ) , thereby having a negative impact on pmn adhesion to fibrinogen , chemotaxis , and phagocytosis .
altogether , these studies suggest that cyth-1 in pmns differentially regulates the activation of the 2 integrins lfa-1 and mac-1 .
gtpase activating proteins ( gaps ) are proteins that accelerate the intrinsic gtp hydrolysis activity of small g proteins .
the arf gaps contain a characteristic domain of about 130 amino acids , which has been shown to be the minimum unit with gap activity .
the gap domain has a zinc - finger structure that is unique to arf gaps , but similar to other gaps for the ras and rho families of small gtpases , there is a conserved arginine that is essential for the catalytic activity of the so - called  arginine finger  [ 125 , 126 ] . the human genome is predicted to encode thirty - one proteins containing the arf gap domain .
mammalian arf gaps are selective for one or more arfs   but are not active on sar1 or arl proteins .
however , gap selectivity in vivo is likely to depend on the localization of arf gaps and arfs in cells , as well as on composition and shape of lipid bilayer membranes .
in addition to the gap domain , these proteins have a variety of other domains involved in intramolecular , protein - protein , and protein - lipid interactions .
the arf gaps have been classified into two major groups according to the domain structure .
the arfgap1-type with n - terminal gap domain includes the arfgap , smap , adap , and git protein subtypes .
the azap - type with a gap domain in a sandwich between a ph domain and the ankyrin ( ank ) repeat motif comprises asap , acap , agap , and arap subtypes .
arfgap1 contains two motifs termed amphipathic lipid packing sensor ( alps ) that allow binding to liposomes . the alps motifs for golgi localization make arfgap1 activity extremely sensitive to membrane lipid curvature [ 132 , 133 ] .
the primary function attributed to arfgap1 is regulation of copi vesicle biogenesis by stimulating the hydrolysis of gtp bound to golgi arfs [ 124 , 134 ] .
arfgap2 and arfgap3 lack the alps motif of arfgap1 but instead possess dilysine retrieval motifs that confer golgi localization through direct interaction with the copi coat [ 135137 ] .
arfgap2 and arfgap3 are key components of the copi coat lattice and coatomer - induced gap activity may be required for proper vesicle formation [ 135137 ] .
the smap subfamily comprises two members , smap1 and smap2 [ 138 , 139 ] .
smap protein structure includes a clathrin box that binds clathrin heavy chains and regulates the trafficking of clathrin - coated vesicles [ 138 , 139 ] .
smap1 was reported to be an arf6 gap regulating arf6-dependent endocytosis of transferrin and e - cadherin receptors , whereas smap2 was involved in arf1-dependent membrane trafficking between early endosomes and the tgn .
although smap1 and smap2 were initially shown to have distinct functions , the proteins were also reported to interact which each other and to regulate transferrin receptor endocytosis .
there are no reports , as of yet , on the expression of smap proteins in human pmns and in human tumor - derived myeloid cell lines .
though git proteins have no ph domain , their gap activity was reported to be stimulated by ptdins(3,4,5)p3 . git1
interacts with various gpcrs to regulate their endocytosis via the clathrin pathway in a g protein - coupled receptor kinase , -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 .
git / pix complexes regulate cdc42/rac - dependent activation of p21-activated kinase 1 ( pak1 ) , a protein involved in microtubule - mediated focal adhesion disassembly .
git2 and a splice variant named git2-short have been characterized , with the expression of the latter being restricted to immune cells .
overexpression of git2-short was reported to cause redistribution of golgi protein -cop , to affect the subcellular localization of paxillin , and to reduce the levels of actin - based fibers .
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 ,
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 .
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 .
more information on the domain structure of asaps can be found elsewhere [ 25 , 127 ] .
asap1 and asap2 have pip2-dependent gap activity and both act on arf1 and arf5 and only weakly on arf6 in vitro .
this is possibly due to intramolecular interaction between the bar and ph domains , which affects gap activity independently of the property of bar domain in mediating association of asap1 with membranes .
although asap1 and asap2 were detected in pmns using proteomic analyses , their subcellular distribution and biological functions remain open questions .
the acap family comprises three members , with acap1 and acap2 being the best characterized .
acap homologs in dictyostelium were shown to affect the actin cytoskeleton and to regulate cytokinesis [ 155 , 156 ] . however , their role in chemotaxis is still unclear [ 154156 ] .
a recent study suggested a role for acap2 in fcr - dependent phagocytosis in macrophages .
polyclonal acap1 antibodies generated in our laboratory detected a protein of about 75  kda in pmns and acap1-gfp overexpressed in rbl-2h3 cells ( figure 3 ) .
but to our knowledge , no one has yet explored the function of this arf gap in pmns .
the three members of the arap subtype have rho gap domain in addition to an arf gap domain with multiple ph domains that recognize ptdins(3,4,5)p3 [ 25 , 127 , 158 ] .
arap1 regulates endocytosis of epidermal growth factor receptor ( egfr ) [ 159 , 160 ] .
receptor internalization requires the interaction of arap1 with multiple proteins such as cin85 and its phosphorylation by src kinase [ 160 , 161 ] .
further investigation is required to assess the impact of phosphorylation and protein - protein interaction on arap1 gap activities .
arap1 also regulated the filamentous - actin ring structure size of circular dorsal ruffles in nih 3t3 cells through an arf1/5-dependent mechanism .
arap2 was shown to regulate focal adhesion dynamics using arf6 . in vitro and in vivo arap3 has been reported to be a specific arf6 gap [ 158 , 164 ] .
arap1 and arap3 were detected in neutrophils using proteomics methods [ 46 , 47 ] .
recent studies using an inducible arap3 ko mouse model suggest that this gap affects 2 integrin functions and several biological responses dependent on integrin activation such as adhesion - dependent ros formation , granule release , and chemotaxis through modulation of rhoa but not of arf6 activation [ 48 , 49 ] . in humans , 11 genes are predicted to encode for agap - type arf gaps , 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 2-adrenergic receptors . during cell migration ,
agap2 was shown to promote focal adhesion disassembly through binding to and stimulation of focal adhesion kinase .
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 ) ) .
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 .
the adap subfamily includes two structurally related proteins with an arf gap and two ph domains in tandem .
adap1 is a brain specific ptdins(3,4,5)p3-binding protein that functions as an arf6 gap in vivo [ 172 , 173 ] .
adap1 also serves as a scaffold in several signalling pathways through interaction with proteins such as f - actin , the kinesin family protein k1f13b , ran binding protein in microtubule organizing center , -tubulin , and pkc family members to name a few ( reviewed in ) . through interaction with components of the cytoskeleton
, adap1 has been suggested to regulate neuronal actin and vesicle transport along microtubules [ 173 , 175 ] .
adap2 is a gap selective for arf6 that regulates cortical actin formation at the plasma membrane .
adap2 is abundantly expressed in fat , heart , and skeletal muscles   and was suggested to play a role in heart development .
the presence of arf proteins including arf1 , arf3 , arf5 , and arf6 has been reported in pmns and/or neutrophil - like cells .
arf1 and arf6 regulate various biological responses through stimulation of the lipid remodelling enzymes pld and pip5-kinase ( table 1 ) .
pharmacological approaches and the use of neutrophil - like cells have permitted the investigation of the role of arf6 in pmn functional responses such as nadph oxidase activity , phagocytosis , and degranulation .
cyth-1 was involved in pld and nadph oxidase activation , degranulation , and regulation of pmn adhesion through the 2 integrins mac-1 and lfa-1 .
gbf1 is part of a signalling pathway coordinating cell polarisation , direction sensing , and superoxide production in response to stimulation with chemoattractants .
there is still fragmentary information available on the biological functions of the various arf gaps expressed by pmns .
the best characterized include git2 , a negative regulator of arf1 , and arap3 , a dual arf and rho gap . whereas git2 is involved in pmn direction sensing and superoxide production , arap3 modulates 2 integrin functions and adhesion - dependent formation of ros , granule content release , and chemotaxis ( table 1 ) .
further studies on the arf gaps recently identified in human pmns ( asap1 , asap2 , acap1 , acap2 , arap1 , adap2 , and agap2 ) are required to understand the significance of these proteins in pmn biology .
 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 . 
1. Introduction
2. Arfs
3. Arf GEFs
4. Arf GAPs
5. Concluding Remarks