Abstract
Abstract Frabin, together with, at least, FGD1, FGD2,
Introduction

Rho family small GTP-binding proteins (G proteins), including
Cdc42, Rac and Rho, regulate the actin cytoskeleton-dependent cellular activities, including cell shape changes, cell migration, cell adhesion and cytokinesis [1, 2] . These Rho family small G proteins also regulate other cellular activities such as the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase cascade, an NADPH oxidase enzyme complex and the transcription factor NF-B [2] . [3] . The Arp2/3 complex interacts with the sides of the pre-existing actin filaments (F-actin) to promote actin polymerization and generate a branched F-actin network. Rho promotes actin polymerization through two distinct targets, p160 and mDia [2] . Despite both Cdc42 and Rac activating the Arp2/3 complex, it is unclear how they induce the formation of morphologically distinct structures, for example, filopodia with straight bundles of F-actin and lamellipodia with branched F-actin. Recent evidence indicates that Cdc42 stimulates actin polymerization through another target protein, mDia2, leading to the formation of filopodia [4] .
Of the actin cytoskeleton-dependent cellular activities in fibroblasts, such as NIH 3T3 and Swiss 3T3 cells, Cdc42 regulates the formation of filopodia, Rac regulates the formation of lamellipodia and ruffles and Rho regulates the formation of stress fibers and focal adhesions [1, 2]. Cdc42 and Rac activate the Arp2/3 complex through their respective target proteins, WiskottAldrich syndrome protein (WASP)/neural (N-)WASP and WASPfamily verprolin-homologous protein (WAVE)
Like other G proteins, the Rho family members have two interconvertible forms: GDP-bound inactive and GTP-bound active forms [1, 2, 5] [1, 5] [7] . FGD1 shows specific GEF activity towards Cdc42 and induces the formation of filopodia and the activation of JNK through the activation of Cdc42 [8] . Subsequently, FGD2 and FGD3 were identified by genetic searches as FGD1 homologues [9, 10] . We isolated frabin, a Cdc42-specific GEF, as an F-actin-binding (FAB) protein showing significant homology to FGD1 [11] . Like FGD1, frabin induces the formation of filopodia through the activation of Cdc42 in fibroblasts [11] [12] [13] . Here, we review frabin and other related Cdc42-specific GEFs.
. Their interconversion is tightly controlled by three types of regulators: guanine nucleotide exchange factors (GEFs) that stimulate the conversion from the GDP-bound form to the GTP-bound form, GDP dissociation inhibitors (GDIs) that inhibit this reaction and GTPase-activating proteins (GAPs) that stimulate the conversion from the GTP-bound form to the GDP-bound form. The modes of action and activation of the Rho family members by these regulators are proposed to be as follows

Molecular structures of frabin and other related GEFs
Frabin, FGD1 [7] , FGD2 [9] , FGD3 [10] (Fig. 1B) .
Frabin binds along the sides of F-actin through the FAB domain [11] . Full [1, 2] . PH domains are found in many molecules and are known for their ability to target cellular membranes by binding to phosphoinositides [16] . PH domains adjacent to the DH domains of several GEFs have been shown to bind specifically to nucleotide-free small G proteins and assist effective guanine nucleotide exchange reactions [17] . Disruption of these interactions between the PH domains and small G proteins by site-directed mutagenesis inhibits GEF activities [18] [12] . Rho GDI inhibits the GEF activity of frabin on Cdc42. A similar fragment of FGD1 also shows Cdc42-specific GEF activity [8] . The [19] . [18] . FGD1 FYVE domain was experimentally shown to recognize both PI3P and phosphatidylinositol 5-phosphate [20] . Thus, frabin and FGDs have not only two PH domains, but also an FYVE domain, and this domain organization suggest that they serve as cross-linkers between the membranes and the actin cytoskeleton.
A large population of PI3P is found in the early endosomes, multi-vesicular bodies and phagosomes. A small population of PI3P is identified in the nucleus, mitochondria, Golgi and plasma membrane. FYVE domains are principally defined by three conserved sequences: the N-terminal WxxD, the central RR/KHHCR and the C-terminal RVC motifs that form a compact PI3P-binding site, but both frabin and FGD1 possess atypical FYVE domains that lack an N-terminal WxxD motif
The amino acid sequence of the DH domain of FRG shows significant homology to those of FGD1 and frabin (Fig. 1B) . Consistently, FRG shows Cdc42-specific GEF activity in a cell-free assay system [14] . [21, 22] . FERM domains bind to both phosphoinositides and the cytoplasmic tails of integral membrane proteins. FRG may also serve as a Cdc42-specific GEF that couples the plasma membrane with the actin cytoskeleton.
FRG consists of an FERM domain, a prolinerich domain, a DH domain and two PH domains, in order, from the N-terminus to the C-terminus. FERM domains, also called band

homology domains, were initially identified in the peripheral membrane proteins that function as cross-linkers between the plasma membrane and F-actin

Tissue distribution, subcellular localization and splicing variants of frabin and other related GEFs
Frabin is expressed in all rat tissues, including the heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis [12] . In cultured rat hippocampal neurons, frabin is highly concentrated at filopodia in the growth cones. Mouse frabin has two smaller splicing variants [23] . The [7, 9, 10, 25] . FGD1 is localized at the subcortical actin cytoskeleton and the Golgi membrane in the osteoblast-like cell line, MC3T3-E1 [26] . It has been shown that Cdc42 and N-WASP are localized together, in part, at the Golgi membrane and that they regulate vesicle trafficking from the Golgi to the ER [4] . FGD1 may be involved in this Golgi-ER transport. FGD1 has also two splicing variants that only possess a proline-rich domain and an incomplete DH domain [27, 28] . These splicing variants may regulate the activation of Cdc42 by competing with the longest type of FGD1. FRG is ubiquitously expressed and localized to cell-cell adhesion sites [14, 29] , but splicing variants have not been reported.
Cellular activities of frabin and other related GEFs
Expression of the full-length frabin induces the formation of filopodia and the activation of JNK through the activation of Cdc42 in fibroblasts [11] [12] [13] (Fig. 2) . Furthermore, the expression of frabin induces the formation of not only filopodia but also lamellipodia in fibroblasts [13] [14] and regulates nectin-and cadherin-based cell-cell adhesion and cell polarity in MDCK epithelial cells [31, 32] . FRG also induces the Cdc42-dependent formation of filopodia and spines in hippocampal neurons [33] .
Roles of the domains of frabin in cellular activities
A fragment of frabin containing the DH domain and the first PH domain shows Cdc42-specific GEF activity in a cell-free assay system, but this fragment does not induce the formation of filopodia or lamellipodia or the activation of JNK in fibroblasts [11] [12] [13] . For the formation of filopodia, the FAB domain is additionally required, suggesting that the association of frabin with the actin cytoskeleton is necessary for this activity (Fig. 2) . A fragment containing the FAB domain is recruited to Cdc42 CA-formed filopodia, but not to Rho CA-formed stress fibers [34] 
. Furthermore, co-expression of this domain inhibits the formation of filopodia induced by the fulllength frabin. These data suggest that the FAB domain competes with the full-length frabin for the association with a specific actin structure(s), and thereby inhibits the formation of filopodia. Because actin structures are determined on the basis of the various FAB proteins they are composed of, it is possible that the association of the FAB domain with the specific actin structures is mediated by interactions not only with F-actin itself, but also with a specific FAB protein(s).
As described above, the expression of Cdc42 CA or Rac CA, or both, does not result in the generation of microspikes in MDCK cells [29] . However, the microspikes are formed when Cdc42 CA is coexpressed with a fragment of frabin minimally including the FAB domain and a mutated DH domain that lacks Cdc42-activating activity [35] .
These data suggest that the region containing the FAB and DH domains directly reorganizes the actin cytoskeleton in a Cdc42-independent manner, and that both the Cdc42-activating and Factin-modulating activities of frabin are required for the generation of microspikes in MDCK cells. Consistently, FGD1 also promotes Cdc-42-independent actin polymerization through its interaction © 2008 The Authors Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd

Fig. 2 Roles of the domains of frabin in cell shape changes. The N-terminal region of frabin containing the FAB domain, the DH domain and the first PH domain induces the formation of filopodia through the direct activation of Cdc42. The C-terminal region of frabin containing the DH domain, the first PH domain, the FYVE domain and the second PH domain induces the formation of lamellipodia through the indirect activation of Rac.
with cortactin [15, 36] [37, 38] . This activity of cortactin has been shown to be enhanced by its interaction with FGD1 [36] .
. Cortactin is a c-Src substrate that binds to and activates the Arp2/3 complex, leading to the formation of a branched F-actin network
On the other hand, the FYVE domain and the second PH domain, in addition to the DH domain and the first PH domain, are necessary for the formation of lamellipodia and the activation of JNK, suggesting that the association of frabin with membranes is required for these activities [12, 13] (Fig. 2) 
Mode of action of frabin in cell shape changes
Cell shape change has long been thought to be determined by the cytoskeleton reorganization beneath the plasma membrane. [3] . It is conceivable that cell shape changes are determined by the synergistic reorganization of the cytoskeleton and the plasma membrane. Therefore, we propose a model for the mode of action of frabin in the formation of filopodia and lamellipodia, as follows: initially, frabin is targeted to a preexisting specific actin structure through its FAB domain (Fig. 3) . Frabin is also recruited to a specific membrane structure(s) through the region including the DH domain, the first PH domain, the FYVE domain and the second PH domain [34] . Like [39] . Frabin may be activated by 3-phosphoinositides in the PI3 kinase signalling pathway.
However, this idea could be revised by the discovery of several membrane-deforming proteins (MDPs), such as the BAR domaincontaining endophilin and EFC domain-containing FBP17, which bind to WASP and WAVE proteins

Mode of activation of frabin and FRG

It is unclear how external or internal stimuli transduce the signals to activate frabin and FGDs; but our recent preliminary results show that some phosphoinositides have a potency to stimulate the GEF activity of frabin. It has also been shown that frabin is recruited by the activation of phosphatidylinositol 3-kinase (PI3 kinase) during parasite invasion
In contrast to frabin and FGDs, the mode of activation of FRG is well studied. FRG is involved in the signalling pathway from the endothelin A receptor to JNK [14] . In this pathway, c-Src phosphorylates FRG and activates Cdc42-specific GEF catalytic activity. The N-terminal region and the DH and PH domains are tyrosinephosphorylated, but it remains unknown as to how this phosphorylation leads to the activation of FRG. FRG is also involved in the signalling pathway underlying the cell adhesion-induced activation of Cdc42 [29, 32] . Cdc42 regulates the formation of cadherinbased adherens and claudin-based tight junctions through actin reorganization in epithelial cells [40, 41] [32, 43] . 4H [24, 45] . CMT 
Involvement of FGD4/frabin and FGD1 in human diseases

Conclusions and perspectives
We have described here our current knowledge of frabin and other related Cdc42-specific GEFs, including FGD1, FGD2, FGD3 

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