Activation of toxin ADP-ribosyltransferases by eukaryotic ADP-ribosylation factors

ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that ate regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (~180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP, of course). Cholera toxin was the first to be recognized, hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking, ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms, which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells, it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain, a sequence of ~200 amino acids that resembles a region in the yeast Sec7 gene product, which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains, among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity, recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4, 5, or 6.     

Structural elements of ADP-ribosylation factor 1 required for functional interaction with cytohesin-1

ADP-ribosylation factor 1 (ARF1) is a 20-kDa guanine nucleotide-binding protein involved in vesicular trafficking. Conversion of inactive ARF-GDP to active ARF-GTP is catalyzed by guanine nucleotide exchange proteins such as cytohesin-1. Cytohesin-1 and its Sec7 domain (C-1Sec7) exhibit guanine nucleotide exchange protein activity with ARF1 but not ARF-like protein 1 (ARL1), which is 57% identical in amino acid sequence. With chimeric proteins composed of ARF1 (F) and ARL1 (L) sequences we identified three structural elements responsible for this specificity. Cytohesin-1 increased [35S]guanosine 5'-(gamma-thio)triphosphate binding to L28/F (first 28 residues of L, remainder F) and to a much lesser extent F139/L, and mut13F139/L (F139/L with random sequence in the first 13 positions) but not Delta13ARF1 that lacks the first 13 amino acids; therefore, a nonspecific ARF N terminus was required for cytohesin-1 action. The N terminus was not, however, required for that of C-1Sec7. Both C-1Sec7 and cytohesin-1 effectively released guanosine 5'-(gamma-thio)triphosphate from ARF1, but only C-1Sec7 displaced the nonhydrolyzable GTP analog bound to mut13F139/L, again indicating that structure in addition to the Sec7 domain is involved in cytohesin-1 interaction. Some element(s) of the C-terminal region is also involved, because replacement of the last 42 amino acids with ARL sequence in F139L decreased markedly the interaction with cytohesin-1. Participation of both termini is consistent with the crystallographic structure of ARF in which the two terminal alpha-helices are in close proximity. ARF1 residues 28-50 are also important in the interaction with cytohesin-1; replacement of Lys-38 with Gln, the corresponding residue in ARL1, abolished the ability to serve as substrate for cytohesin-1 or C-1Sec7. These studies have defined multiple structural elements in ARF1, including switch 1 and the N and C termini, that participate in functional interactions with cytohesin-1 (or its catalytic domain C-1Sec7), which were not apparent from crystallographic analysis.     

Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1)

Activation of ADP-ribosylation factors (ARFs) is mediated by guanine nucleotide-exchange proteins, which accelerate conversion of inactive ARF-GDP to active ARF-GTP. ARF domain protein (ARD1), a 64-kDa GTPase with a C-terminal ADP-ribosylation factor domain, is localized to lysosomes and the Golgi apparatus. When ARD1 was used as bait to screen a human liver cDNA library using the yeast two-hybrid system, a cDNA for cytohesin-1, a approximately 50-kDa protein with ARF guanine nucleotide-exchange protein activity, was isolated. In this system, ARD1-GDP interacted well with cytohesin-1 but very poorly with cytohesin-2. In agreement, cytohesin-1, but not cytohesin-2, markedly accelerated [(35)S]guanosine 5'-3-O-(thio)triphosphate binding to ARD1. The effector region of the ARF domain of ARD1 appeared to be critical for the specific interaction with cytohesin-1. Replacement of single amino acids in the Sec7 domains of cytohesin-1 and -2 showed that residue 30 is critical for specificity. In transfected COS-7 cells, overexpressed ARD1 and cytohesin-1 were partially colocalized, as determined by confocal fluorescence microscopy. It was concluded that cytohesin-1 is likely to be involved in ARD1 activation, consistent with a role for ARD1 in the regulation of vesicular trafficking.     

Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors.

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at 相似文献   

The Arl4 family of small G proteins can recruit the cytohesin Arf6 exchange factors to the plasma membrane

The small GTPase Arf6 regulates endocytosis, actin dynamics, and cell adhesion, and one of its major activators is the exchange factor Arf nucleotide-binding site opener (ARNO), also called cytohesin-2 [1, 2]. ARNO must be recruited from the cytosol to the plasma membrane in order to activate Arf6, and in addition to a Sec7 nucleotide-exchange domain it contains a C-terminal pleckstrin homology (PH) domain that binds phosphoinositides [3, 4]. ARNO and its three relatives, cytohesin-1, Grp1/cytohesin-3, and cytohesin-4, are expressed as two splice variants, with either two or three glycines in a loop in the phosphoinositide-binding pocket of the PH domain [5, 6]. The diglycine form binds PtdIns(3,4,5)P(3) with high affinity and mediates recruitment of cytohesins to the plasma membrane in response to insulin and growth factors [7, 8]. However, the triglycine form has only micromolar affinity for both PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2), affinities that are insufficient to confer membrane recruitment, raising the question of how the triglycine forms of cytohesins are regulated [5, 9]. Here we show that three related Arf-like GTPases of unknown function, Arl4a, Arl4c, and Arl4d, are able to recruit ARNO and other cytohesins to the plasma membrane by binding to their PH domains irrespective of whether they are in the diglycine or triglycine form. The Arl4 family thus defines a signal-transduction pathway that can mediate the plasma-membrane recruitment of cytohesins independently of a requirement for the generation of PtdIns(3,4,5)P(3).     

Phosphoinositides determine specificity of the guanine-nucleotide exchange activity of cytohesin-1 for ADP-ribosylation factors derived from a mammalian expression system.

ADP-ribosylation factors (ARFs) are small Ras-like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin-1, a 47-kDa cytoplasmic protein acts as an inside-out signaling molecule and regulates binding of the beta2 integrin leukocyte function antigen 1 (LFA-1) to its ligand intercellular adhesion molecule 1 (ICAM-1). In this study, we address the regulation of the GEF activity of cytohesin-1 by phosphoinositides, using mammalian expression of functional ARF-Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A-Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro. We show that both ARF1-Ig and ARF6-Ig chimeras are activated in vitro by cytohesin-1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3). In contrast, cytohesin-1-dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin-1.     

ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling

ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling.     

ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1.

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.     

The Guanine Nucleotide Exchange Protein for ADP-ribosylation Factor 6, ARF-GEP100/BRAG2, Regulates Phagocytosis of Monocytic Phagocytes in an ARF6-dependent Process

Phagocytosis is a complex multistep process requiring diverse signaling and regulatory molecules. ADP-ribosylation factor 6 (ARF6), a small GTPase, is known to regulate membrane trafficking and the actin cytoskeketon at the plasma membrane and functions as a regulatory molecule of phagocytosis. ARF activity is regulated by cycling between GDP-bound and GTP-bound forms. ARF activation is catalyzed by guanine nucleotide exchange factors (GEFs) that facilitate GTP binding. We had earlier reported a 100-kDa ARF-GEF, termed ARF-guanine nucleotide exchange protein 100, GEP100, that preferentially activates ARF6 and was also described by Dunphy et al. (Dunphy, J. L., Moravec, R., Ly, K., Lasell, T. K., Melancon, P., and Casanova, J. E. (2006) Curr. Biol. 16, 315–320) as brefeldin A-resistant ARF-GEF2 (BRAG2). We have now examined a role for GEP100 in phagocytosis. Stable depletion of GEP100 decreased phagocytosis of serum-treated zymosan and IgG-coated latex beads by human monocyte-macrophage-like U937 cells differentiated with phorbol 12-myristate 13-acetate. Decrease of phagocytic activity by RNAi was not rescued by GEP100ΔSec7, a deletion mutant lacking the ARF-activating domain. GEP100-depleted cells also exhibited reduced F-actin fibers around internalized particles. Attachment of these particles to cells and amounts of C3bi and Fcγ receptors, however, were not affected by GEP100 depletion. On immunofluorescence microscopy, GEP100 and ARF6 were concentrated and partially colocalized around internalized particles. Phagocytosis by GEP100-depleted cells was not further affected by depletion of ARF6. Phagocytic activity of GEP100-depleted cells was, however, rescued by expression of the constitutively active ARF6Q67N mutant but not by the dominant-negative ARF6T27N mutant. These data are consistent with the conclusion that GEP100 functions in phagocytosis via its role in ARF6-dependent actin remodeling.     

Signaling by human herpesvirus 8 kaposin A through direct membrane recruitment of cytohesin-1

The induction of a transformed cellular phenotype by viruses requires the modulation of signaling pathways through viral proteins. We show here that the phenotypic changes induced by the kaposin A protein of human herpesvirus 8 are mediated through its direct interaction with cytohesin-1, a guanine nucleotide exchange factor for ARF GTPases and regulator of integrin-mediated cell adhesion. Focus formation, stress fiber dissolution, and activation of the ERK-1/2 MAP kinase signal cascade were reverted by the cytohesin-1 E157K mutant, which is deficient in catalyzing guanine nucleotide exchange. Furthermore, liposome-embedded kaposin A specifically stimulates cytohesin-1 dependent GTP binding of myristoylated ARF1 in vitro. These results suggest a previously unknown involvement of ARF GTPases in the control of cellular functions by herpesviruses.     

Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors

ARF proteins are important regulators of membrane dynamics and protein transport within the eukaryotic cell. The Sec7 domain is approximately 200 amino acids in size and stimulates guanine-nucleotide exchange on members of the ARF class of small GTPases. The members of one subclass of Sec7-domain proteins are direct targets of the secretion-inhibiting drug brefeldin A, which blocks the exchange reaction by trapping a reaction intermediate in an inactive, abortive complex. A separate subclass of Sec7-domain proteins is involved in signal transduction and possess a domain that mediates membrane binding in response to extracellular signals.     

Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain

We demonstrate that the major in vivo targets of brefeldin A (BFA) in the secretory pathway of budding yeast are the three members of the Sec7 domain family of ARF exchange factors: Gea1p and Gea2p (functionally interchangeable) and Sec7p. Specific residues within the Sec7 domain are important for BFA inhibition of ARF exchange activity, since mutations in these residues of Gea1p (sensitive to BFA) and of ARNO (resistant to BFA) reverse the sensitivity of each to BFA in vivo and in vitro. We show that the target of BFA inhibition of ARF exchange activity is an ARF-GDP-Sec7 domain protein complex, and that BFA acts to stabilize this complex to a greater extent for a BFA-sensitive Sec7 domain than for a resistant one.     

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H⁺-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe⁵, Met¹⁰, and Gln¹⁴ involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.     

B2-1, a Sec7- and pleckstrin homology domain-containing protein, localizes to the Golgi complex

B2-1 is a human protein that contains both a Sec7 and a pleckstrin homology domain. The yeast Sec7 protein was previously shown to be involved in vesicle formation in the Golgi and endoplasmic reticulum. Recently, several groups have shown that B2-1 and highly similar proteins (e.g., ARNO, ARNO3) have varied cellular functions and subcellular locations. One of these is an association of the B2-1 Sec7 domain with the plasma membrane, binding to the cytoplasmic portion of the integrin beta2 chain (CD18) and is postulated to be involved in inside-out signaling. Other groups have shown that B2-1 and these related proteins are guanine nucleotide-exchange factors that act upon ADP ribosylation factors (ARFs) and are localized to the Golgi or plasma membrane. Here we report the subcellular localization of B2-1 protein. Interestingly, B2-1 does not localize to the plasma membrane but rather associates with a distinct Golgi complex compartment. B2-1's distribution can be disrupted by brefeldin A, a drug that rapidly disrupts the Golgi apparatus by inhibiting ARF activity. Furthermore, transient transfection of GFP-tagged B2-1 shows Golgi complex targeting. Excessive overexpression of transfected B2-1 causes partial Golgi dispersion.     

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ～200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.     

Cytohesin-1 is a dynamic regulator of distinct LFA-1 functions in leukocyte arrest and transmigration triggered by chemokines

Cytohesin-1 is a regulatory interaction partner of the beta2 integrin alphaLbeta2 (LFA-1) and a guanine exchange factor (GEF) for ADP ribosylation factor (ARF)-GTPases. However, a functional role of cytohesin-1 in leukocyte adhesion to activated endothelium and subsequent transmigration in response to chemokines has not been defined. Overexpression of cytohesin-1 increased LFA-1-dependent arrest of leukocytic cells triggered by chemokines on cytokine-activated endothelium in flow while reducing the fraction of rolling cells. Conversely, a dominant-negative PH domain construct of cytohesin-1 but not a mutant deficient in GEF activity impaired arrest, indicating an involvement of the PH domain while GEF function is not required. Expression of these constructs and a beta2 mutant interrupting the interaction with cytohesin-1 indicated that shape change in flow and transendothelial chemotaxis involve both LFA-1 avidity regulation and GEF activity of cytohesin-1. As a potential downstream target, ARF6 but not ARF1 was identified to participate in chemotaxis. Our data suggest that cytohesin-1 and ARF6 are involved in the dynamic regulation of complex signaling pathways and cytoskeletal remodeling processes governing LFA-1 functions in leukocyte recruitment. Differential effects of cytohesin-1 and ARF6 mutants in our systems reveal that cytohesin-1 with its GEF activity controls both conversion of rolling into firm arrest and transmigration triggered by chemokines, whereas a cyclical activity of ARF6 plays a more important role in diapedesis.     

Getting the right cells to the array: Gene expression microarray analysis of cell mixtures and sorted cells.

BACKGROUND: Most biological samples are cell mixtures. Some basic questions are still unanswered about analyzing these heterogeneous samples using gene expression microarray technology (MAT). How meaningful is a cell mixture's overall gene expression profile (GEP)? Is it necessary to purify the cells of interest before microarray analysis, and how much purity is needed? How much does the purification itself distort the GEP, and how well can the GEP of a small cell subset be recovered? METHODS: Model cell mixtures with different cell ratios were analyzed by both spotted and Affymetrix MAT. GEP distortion during cell purification and GEPs of purified cells were studied. CD34+ cord blood cells were purified and analyzed by MAT. RESULTS: GEPs for mixed cell populations were found to mirror the cell ratios in the mixture. Over 75% pure samples were indistinguishable from pure cells by their overall GEP. Cell purification preserved the GEP. The GEPs of small cell subsets could be accurately recovered by cell sorting both from model cell mixtures and from cord blood. CONCLUSIONS: Purification of small cell subsets from a mixture prior to MAT is necessary for meaningful results. Even completely hidden GEPs of small cell subpopulations can be recovered by cell sorting.     

The N-terminal coiled coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with the scaffolding protein CASP

Cytohesin is a guanine nucleotide exchange factor that regulates members of the ADP-ribosylation factor (ARF) family of small GTPases. All of the members of the cytohesin family (including ARNO, ARNO3, and the newly characterized cytohesin-4) have a similar domain distribution consisting of a Sec7 homology domain, a pleckstrin homology domain, and an N-terminal coiled coil. In this study, we attempt to identify proteins that interact specifically with the coiled coil motif of cytohesin. Yeast two-hybrid screening of a B cell library using the cytohesin N terminus as bait, identified CASP, a scaffolding protein of previously unknown function, as a binding partner. CASP contains an internal coiled coil motif that is required for cytohesin binding both in vitro and in COS-1 cells. The specificity of the coiled coil of CASP is not restricted to cytohesin, however, because it is also capable of interacting with other members of the cytohesin/ARNO family, ARNO and ARNO3. In immunofluorescence experiments, CASP localizes to perinuclear tubulovesicular structures that are in close proximity to the Golgi. These structures remain relatively undisturbed when the cells are treated with brefeldin A. In epidermal growth factor-stimulated COS-1 cells overexpressing cytohesin and CASP, cytohesin recruits CASP to membrane ruffles, revealing a functional interaction between the two proteins. These observations collectively suggest that CASP is a scaffolding protein that facilitates the function of at least one member of the cytohesin/ARNO family in response to specific cellular stimuli.