Dynamic reorganization of the actin cytoskeleton is essential for many cellular activities, such as cell shape changes, cell migration, cell adhesion, and cytokinesis. The Rho family small GTP-binding proteins (G proteins), including Cdc42, Rac, and Rho, regulate these actin cytoskeleton-dependent cellular activities (Takai et al. 2001; Hall 2005). 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. Cdc42 and Rac activate the Arp2/3 complex through their respective target proteins, Wiskott-Aldrich syndrome protein (WASP)/neural (N-)WASP and WASP-family verprolin-homologous protein (WAVE) (Takenawa and Suetsugu 2007). The Arp2/3 complex interacts with the sides of preexisting 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 (Hall 2005). In addition to these actin cytoskeleton-dependent activities, the Rho family small G proteins regulate other cellular activities, such as the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) cascade, an NADPH oxidase enzyme complex, and the transcription factor NF-κB (Hall 2005).
The Rho family members cycle between the GDP-bound inactive and GTP-bound active forms (Takai et al. 2001; Hall 2005). This cycling is tightly controlled by three types of regulators: guanine nucleotide exchange factors (GEFs) that stimulate conversion from the GDP-bound form to the GTP-bound form; GDP dissociation inhibitors that inhibit this reaction; and GTPase activating proteins that stimulate conversion from the GTP-bound form to the GDP-bound form. Most GEFs for the Rho family members share two conserved domains: a Dbl homology (DH) domain of about 250 amino acids, for which the Dbl oncogene product is the prototype, and a pleckstrin homology (PH) domain of about 100 amino acids. Many GEFs for the Rho family members were originally identified as oncogenes (Takai et al. 2001; Hall 2005), but FGD1, which encodes a GEF specific to Cdc42, was discovered by positional cloning to be the gene responsible for faciogenital dysplasia (FGDY) (Pasteris et al. 1994). FGD1 homologs were subsequently identified: FGD2, FGD3, FGD5, and FGD6 were identified by genetic searches; in contrast, frabin/FGD4 was identified by protein purification as an F-actin-binding protein (Obaishi et al. 1998; Nakanishi and Takai 2008).
Molecular Structure, Tissue Distribution, and Splicing Variants
DH domains encode a principal GEF catalytic unit and are required to stimulate GDP release from the Rho family small G proteins (Takai et al. 2001; Hall 2005). PH domains not only serve to target cellular membranes by binding to phosphoinositides but also participate in the binding of small G proteins to facilitate guanine nucleotide exchange reactions (Rossman et al. 2002; Lemmon 2004). The amino acid sequence of the DH domain and the first PH domain of frabin shows significant homology to those of FGD1, FGD2, FGD3, FGD5, and FGD6 (Nakanishi and Takai 2008). A fragment of frabin containing the DH domain and the first PH domain stimulates the guanine nucleotide exchange reaction of Cdc42 in a cell-free assay system (Umikawa et al. 1999). This GEF activity is specific to Cdc42. The frabin fragment does not show the GEF activity toward Rho or Rac.
FYVE domains have been found in many proteins involved in membrane trafficking and phosphoinositide metabolism, and have been shown to specifically interact with one of phosphoinositides, phosphatidylinositol 3-phosphate (PI3P) (Kutateladze 2006). PI3P is found on the limiting membrane domain of early endosomes. However, frabin as well as FGD1 possesses an atypical FYVE domain, which recognizes not only PI3P but also another phosphoinositide, phosphatidylinositol 5-phosphate (PI5P) (Sankaran et al. 2001). PI5P is proposed to be present on late endosomes and at the plasma membrane (Lecompte et al. 2008). It is, therefore, likely that the two PH and FYVE domains of frabin serve to target to specific membrane domains or structures by binding to phosphoinositides.
Rat frabin is expressed in all the tissues, including heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis (Umikawa et al. 1999). In cultured rat hippocampal neurons, frabin is highly concentrated at filopodia in growth cones. Mouse frabin has two smaller splicing variants (Ikeda et al. 2001a). The original biggest, middle, and smallest variants are named frabin-α, -β, and -γ, respectively. Frabin-β lacks the second PH domain, whereas frabin-γ lacks the FYVE domain and the second PH domain. These three splicing variants are expressed in all the tissues, but their expression levels differ among tissues. In this entry, unless otherwise indicated, frabin represents the α form. Human frabin has other splicing variants, one of which is deprived of the FAB domain (Delague et al. 2007). The splicing variants of frabin induce partly different morphological changes, suggesting that the variants may have different physiological functions (Ikeda et al. 2001a).
Role of Domains in Cellular Activities
In fibroblasts, exogenous expression of full-length frabin induces the formation of filopodia and the activation of JNK through the activation of Cdc42 (Obaishi et al. 1998; Umikawa et al. 1999; Ono et al. 2000). The DH domain and the first PH domain are sufficient for the activation of Cdc42 in a cell-free assay system, but these two domains alone do not induce the formation of filopodia or the activation of JNK in fibroblasts (Obaishi et al. 1998; Umikawa et al. 1999). The FAB domain is additionally required for the formation of filopodia, suggesting that the association of frabin with the actin cytoskeleton is necessary for the formation of filopodia (Fig. 1). The FAB domain is recruited to the constitutively active mutant (CA) of Cdc42-formed filopodia, but not to Rho CA-formed stress fibers (Kim et al. 2002). Furthermore, coexpression of this domain inhibits the formation of filopodia induced by full-length frabin. It is likely that the FAB domain competes with full-length frabin for the association with a specific actin structure(s), and thereby inhibits the formation of filopodia.
Expression of frabin induces the formation of not only filopodia, but also lamellipodia, in fibroblasts (Ono et al. 2000) (Fig. 1). This morphological change is inhibited by a dominant-negative mutant (DN) of Rac, indicating that the formation of lamellipodia is mediated by the activation of Rac. 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 (Umikawa et al. 1999; Ono et al. 2000) (Fig. 1). Expression of the fragment containing the mutated DH, first PH, FYVE, and second PH domains inhibits the formation of membrane ruffles induced by full-length frabin; however, the expression of shorter fragments, such as the FYVE domain alone, does not result in this inhibitory action. The mutated DH domain is constructed to lack the GEF activity. It is, therefore, likely that this fragment, containing the DH, first PH, FYVE, and second PH domains, competes with full-length frabin for the association with a specific membrane structure(s). The highly ordered structure of this fragment may be required for its interaction with the specific membrane structure(s), because shorter fragments, such as the FYVE domain alone, do not show a DN effect.
In epithelial cells, such as MDCK cells, frabin induces the formation of microspikes at the basal area of the lateral membrane through the activation of both Cdc42 and Rac, although Cdc42 CA alone, Rac CA alone, or both do not induce the formation of microspikes (Yasuda et al. 2000). However, microspikes are formed when Cdc42 CA is coexpressed with a fragment of frabin minimally including the FAB domain and a mutated DH domain, which lacks the GEF activity (Ikeda et al. 2001b). 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-GEF and F-actin-modulating activities of frabin are required for the generation of microspikes in MDCK cells.
Possible Mode of Action in Morphological Changes
A model for the mode of action of frabin in the formation of filopodia and lamellipodia is proposed as follows: initially, frabin is targeted to a preexisting specific actin structure through the FAB domain. Once recruited, frabin reorganizes the actin cytoskeleton through the action of its N-terminal region, including the FAB domain and the DH domain, in a Cdc42-independent manner. In addition, frabin activates Cdc42 through the DH domain and the first PH domain in the vicinity of the actin structure(s), resulting in the WASP/N-WASP-induced generation of branched F-actin. The F-actin-bundling activity of frabin may contribute to the formation of bundled F-actin in filopodia. Furthermore, Cdc42 stimulates actin polymerization via mDia2 (Hall 2005). The cooperation of Cdc42-independent and Cdc42-dependent actin reorganization finally induces the formation of filopodia. This newly formed actin structure further recruits frabin in a positive feedback cycle to lengthen the filopodia. 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 (Kim et al. 2002). Frabin activates Rac in the vicinity of membrane structure(s), resulting in the WAVE-induced generation of branched F-actin. WAVE furthermore induces outward membrane protrusion at the plasma membrane (Takenawa and Suetsugu 2007). The synergistic reorganization of the actin cytoskeleton and the plasma membrane finally induces the formation of lamellipodia.
Involvement in Human Diseases
Many pathogen microbes, including viruses, bacteria, and parasites, utilize the host-cell actin cytoskeleton for multiple actions, such as attachment, entry into cells, and movement within and between cells (Gruenheid and Finlay 2003). Cryptosporidium parvum, an intracellular parasite, is one of the most commonly reported enteric pathogens worldwide. Frabin was shown to mediate the cellular invasion of this parasite (Chen et al. 2004). C. parvum recruits phosphoinositide 3-kinase to the host cell–parasite interface, an event that then results in the recruitment of frabin, leading to the activation of Cdc42 and subsequent host-cell actin reorganization during cellular invasion.
Mutations in human frabin have been identified to be responsible for the Charcot–Marie–Tooth (CMT) disorder Type 4H (Delague et al. 2007; Stendel et al. 2007). CMT disorders are clinically and genetically heterogeneous hereditary motor and sensory neuropathies characterized by muscle weakness and wasting, foot and hand deformities, and electrophysiological changes. The CMT4H subtype is an autosomal recessive demyelinating form of CMT disorder. Patients show early disease onset, but slowly progressive sensorimotor neuropathy. Nerve biopsy specimens display a severe loss of myelinated fibers, thinly myelinated axons, and outfolding of myelin sheaths. These data indicate that frabin plays an important role in the proper myelination of the peripheral nervous system.
Frabin/FGD4, together with at least FGD1, FGD2, FGD3, FGD5, and FGD6, is a member of a family of Cdc42-specific GEFs. Frabin consist of FAB, DH, first PH, FYVE, and second PH domains, in order, from the N-terminus to the C-terminus. Frabin associates with specific actin and membrane structures and activates Cdc42 and Rac in the vicinity of these structures, resulting in the reorganization of the actin cytoskeleton coupled with membrane dynamics. Thus, it has been shown how frabin induces cell shape changes. However, important questions still remain to be solved: (1) how external stimuli and intracellular signals induce the activation of frabin; (2) the physiological significance of a variety of splicing variants; (3) how frabin induces the activation of Rac; and (4) how frabin regulates myelination. Solving these questions will lead to a better understanding of the modes of action and activation of frabin.
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