The small GTPase ARF serves as a key regulator of a number of cellular processes including vesicle trafficking, signal transduction and regulation of the actin cytoskeleton 
. The six isoforms of ARF in mammals can be categorized into three classes: Class I (ARF1, 2 and 3), Class II (ARF4 and 5) and Class III (ARF6). Among them, ARF1, the most extensively studied Class I member, regulates the formation of coated vesicles on secretory and endocytic membranes 
and also take part in signal transduction in a number of signaling pathways 
. ARF6 functions in the endocytic pathway and regulates actin cytoskeleton 
. Because of their diverse and complex cellular functions, the activity of ARF proteins is highly regulated. Like other small GTPases, ARFs cycle between an active, GTP-bound form and an inactive, GDP-bound form. The intrinsic GTPase activity of ARF is negligible 
and the inactivation of ARF requires interactions with G
roteins (ARF GAPs). All ARF GAPs have an ARF GAP catalytic domain of 120 amino acids enriched in cysteine. To date, there are at least 24 such sequences identified in the human genome 
and the genes containing them are divided into 10 subfamilies. With the exception of the ADAP subfamily, at least one member within all other subfamilies have been demonstrated experimentally to have ARF GAP activity 
. Other than the ARF GAP catalytic domain, these genes are vastly different in terms of their size, the identifiable features that they contain and their subcellular localization 
. The existence of multiple domains in these molecules not only reflects the complex functions of ARF but also indicates that these ARF-GAPs have functions in addition to stimulating GTP hydrolysis on ARF.
ARFGAP1 was the first ARF-GAP protein isolated 
. This protein contains 415 amino acids, with a GAP catalytic domain located in the amino terminal 120 residues. ARFGAP1 was shown to localize primarily on Golgi membranes where it is thought to function in the formation of coated vesicles 
. The non-catalytic sequences of ARFGAP1 are necessary for associating with membranes. We have identified a region in the middle of the protein that is responsible for the membrane targeting of the whole molecule both in vivo
and in vitro 
. This region contains 132 residues starting from amino acid 203 to 334. Subsequently, a lipid-package sensor domain that allows the protein to sense membrane curvature was discovered in this region of the molecule 
Gcs1p is the ARF GAP in S. cerevisiae with the greatest sequence similarity to ARFGAP1 in its non-catalytic sequences. Besides its anticipated functions in the Golgi 
, Gcs1p is also required for normal organization of the actin cytoskeleton 
interacts genetically with SLA2
. Mutants in the SLA2
gene are defective in actin polarization and endocytosis 
encodes an actin cross-linking protein, homologous to human fimbrin. In addition to this genetic evidence for a role for GCS1 in regulating actin, Gcs1p stimulates actin polymerization and inhibits depolymerization in vitro 
The Rho family of small GTPases, consisting of Cdc42, Rac and Rho, are the key regulators of the actin cytoskeleton 
. Each of these small GTPases stimulates the formation of a unique type of actin morphology, although their activities are often coordinated. For example, Cdc42 stimulates the production of actin microspikes or filopodia 
. Rac1 stimulates the formation of lamellipodia and membrane ruffles 
. RhoA stimulates the formation of stress fibers and focal adhesions 
. ARF6 has also been demonstrated to regulate the actin cytoskeleton. Earlier cell biology studies using GTP-binding mutants of ARF6 has demonstrated the important function that ARF6 regulates the reorganization of cortical actin 
. ARF6 appears to exert its effect on the cortical actin cytoskeleton by acting coordinately with Rac1 
. ARF6 activates Rac by interacting with Rac GEFs or by regulating the availability of lipid rafts required for Rac attachment at the plasma membrane.
Of note, a subfamily of the ARF GAPs, called ARAP1, 2 and 3, have been shown to act as GAPs for both the ARF family small GTPases and the Rho family small GTPases Cdc42 and RhoA 
. ARAP1 is primarily localized on Golgi membranes, similar to ARFGAP1, suggesting that it might function in Golgi-derived vesicle trafficking. The presence of both an ARF GAP and a RhoGAP domain in ARAP proteins suggests that Class I ARFs and Rho proteins might interact functionally, as has been demonstrated for ARF6.
While studying the vesicle trafficking functions of ARFGAP1 we noticed that in cells overexpressing GAP273, a truncated protein containing only the carboxyl terminal 273 residues (residues 142–415) of ARFGAP1, cell spreading and flattening appeared to be impaired. Because the closest homologue of ARFGAP1 in yeast, Gcs1p, regulates the actin cytoskeleton as well as membrane traffic, we investigated the potential effect of ARFGAP1 on actin. We found that ARFGAP1, besides being localized on Golgi membranes, is present in the cell periphery associated with actin foci. We observed that cells over-expressing GFP-GAP273 are slow to spread when plated on fresh culture dishes. This effect is mediated by the carboxyl terminal 65 residues of ARFGAP1, and is related to the function of Rac1, as the over-expression of GAP273 inhibits the activation of Rac1.