Yeast two-hybrid screening of a human kidney cDNA library using the GTP-bound form of a class II ADP-ribosylation factor (ARF5) identified a novel ARF5-binding protein with a calculated molecular mass of 82.4 kDa, that was named arfophilin. Northern hybridization analysis showed high level arfophilin mRNA expression in human heart and skeletal muscle. Arfophilin bonds only to the active, GTP-bound form of ARF5 and does not bind to GTP-ARF3, which is a class I ARF. The N terminus of ARF5 (1-17 amino acids) is essential for binding to arfophilin. The GTP-bound form of ARF5 with amino acid residues in the N terminus mutated to those in ARF4 (another class II ARF) also binds to arfophilin, suggesting it is a target protein for GTP-bound forms of class II ARFs. The binding site for ARF on arfophilin is localized to the C terminus (residues 612-756), which contains putative coiled-coil structures. Recombinant arfophilin overexpressed in CHO-K1 cells is localized in the cytosol and translocates to a membrane fraction in association with GTP-bound ARF5. ARF5 containing the N terminus of ARF3 does not promote translocation, indicating that class II ARFs are specific carriers for arfophilin (Shin, 1999).
Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5 (amino acids 2-17), which is distinct from that of class I or class III ARFs, is essential for binding to the C-terminus of arfophilin (amino acids 612-756). Using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates it has been shown that, unexpectedly, ARF6 also binds to full-length arfophilin or the C-terminus of arfophilin (amino acids 612-756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further show that the amino acid sequence of residues 37-80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, is essential for binding to arfophilin. Both GTP-ARF5 and GTP-ARF6 bind to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 does not bind. In contrast, all three forms of ARF bind to arfaptin 2, with ARF1 showing the strongest binding. Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 is autoactivating. These findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector (Shin, 2001).
Rab11 (see Drosophila Rab-protein 11), a low molecular weight GTP-binding protein, has been shown to play a key role in a variety of cellular processes, including endosomal recycling, phagocytosis, and transport of secretory proteins from the trans-Golgi network. A novel Rab11 effector, EF-hands-containing Rab11-interacting protein (Eferin), is described in this study. In addition, a 20-amino acid domain has been defined that is present at the C terminus of Eferin and other Rab11/25-interacting proteins, such as Rip11 and nRip11. Using biochemical techniques, this domain is shown to be necessary and sufficient for Rab11 binding in vitro and it is required for localization of Rab11 effector proteins in vivo. The data suggest that various Rab effectors compete with each other for binding to Rab11/25 possibly accounting for the diversity of Rab11 functions (Prekeris, 2001).
Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling system. It has been hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. A family of four Rab11-interacting proteins is defined in this study: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family Interacting Protein 2 (Rab11-FIP2), Rab11-Family Interacting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associate with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacts with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a is dependent upon a conserved carboxyl-terminal amphipathic alpha-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalize with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalizes with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also co-enrich with Rab11a and H(+)K(+)-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocate with Rab11a and the H(+)K(+)-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors (Hales, 2001).
Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. A novel 80-kDa protein has been identified that interacts specifically with the GTP-bound conformation of Rab4, and it also interacts strongly with Rab11. This protein has been named Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homolog of Rip11 and therefore belongs to the recently described Rab11-FIP family (Lindsay, 2002a).
Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells. The specificity of the interaction between Rab11-FIP2 and Rab11 has been characterized; it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. The COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself is insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, causes tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 is also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking (Lindsay, 2002b).
The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Several of these proteins have been characterized with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. Two of the family members-pp75/Rip11 and Rab11-FIP3 do not bind Rab4; several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end that includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. A new (sixth) member of the protein family has been identified, that has been named Rab11-FIP4; the family evolutionary complexity and chromosomal distribution is reported. Furthermore, it is proposed that the ability of these proteins to bind each other are important in effecting membrane trafficking events by forming protein 'platforms,' regulated by Rab11 and/or Rab4 activity (Wallace, 2002a).
Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and its C-terminal region allows the protein to self-interact and interact with pp75/Rip11, Rab11-FIP2, and Rab11-FIP3. However, Rab11-FIP4 does not appear to interact directly with Rab coupling protein (RCP). The subcellular localization of Rab11-FIP4 in HeLa cells was investigated; it colocalizes extensively with transferrin and with Rab11. Furthermore, when overexpressed, it causes a condensation of the Rab11 compartment in the perinuclear region. The carboxy-terminal region of Rab11-FIP4 [Rab11-FIP4(C-ter)] is necessary and sufficient for its endosomal membrane association. Expression of Rab11-FIP4(C-ter) causes a dispersal of the Rab11 compartment towards the cell periphery and does not inhibit transferrin recycling in HeLa cells. It is likely that Rab11-FIP4 serves as a Rab11 effector in a Rab11 mediated function other than transferrin recycling (Wallace, 2002b).
Rab11-FIP2 is a member of a newly identified family of Rab11-binding proteins that have been implicated in the function of recycling endosomes. Rab11-FIP2 may also be involved with the process of receptor-mediated endocytosis. Rab11-FIP2 contains an NPF motif that allows it to bind Reps1, a member of a family of EH domain proteins involved in endocytosis. Rab11-FIP2 associates with the alpha-adaptin subunit of AP-2 complexes, which are known to recruit receptors into clathrin-coated vesicles. Overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors, but not transferrin receptors, through binding sites that promote complex formation with Rab11, Reps1, and alpha-adaptin. These findings suggest that Rab11-FIP2 may participate in the coupling of receptor-mediated endocytosis to the subsequent sorting of receptor-containing vesicles in endosomes (Cullis, 2002).
Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and it binds both Arf5 and Rab11. A related protein, arfophilin-2, is described that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localizes to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment is selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions are localized around the centrosome in a region that is also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment is altered. The arfophilins belong to a conserved family that includes Drosophila Nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-Nuclear fallout in HeLa cells results in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. It is suggested that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment (Hickson, 2003).
The observation that GFP-Nuf overexpression in HeLa cells results in largely the same phenotype as that of GFP-arfophilin-2 overexpression suggests that these proteins are functionally related. This has several important implications. It suggests a potential role for the arfophilins in cell division, particularly cytokinesis, because Nuf is required for the formation of cellularization furrows in the Drosophila embryo. Because testis is a tissue undergoing a high degree of cell division, such a role may help to explain the high level of expression observed in this tissue. The findings also implicate the endosomal recycling compartment in cytokinesis. This is consistent with recent studies in C. elegans where RNA interference-induced suppression of Rab 11 leads to specific regression of the cleavage furrow at the final stage of abscission (Skop, 2001). The significance of the role of Rab 11 should not be overlooked, particularly given the interaction of arfophilins with this GTPase. Arfs have also been implicated in cellularization in Drosophila and cytokinesis in C. elegans. Such observations suggest the hypothesis that arfophilin-2, and by extension the endosomal recycling compartment, may be involved in traffic to the midbody during cytokinesis. Moreover, the identification of arfophilin-2 as a Rab 11 and Arf binding protein offers the tantalizing suggestion that this protein may integrate the Rab 11 and Arf signals to membrane traffic during cytokinesis (Hickson, 2003 and references therein).
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