Interaction of GRASP, a Protein encoded by a Novel Retinoic Acid-induced Gene, with Members of the Cytohesin Family of Guanine Nucleotide Exchange Factors

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.

Section: Casp Intracellular Localization Is Perinuclear In Cos-1 Cells-mentioning

confidence: 99%

mentioning

confidence: 99%

The N-terminal Coiled Coil Domain of the Cytohesin/ARNO Family of Guanine Nucleotide Exchange Factors Interacts with the Scaffolding Protein CASP

Mansour

Lee

Pohajdak

2002

“…Some cytohesin family binding proteins have been identified and characterized. The coiled-coil domain provides a binding site with other coiled-coil domain-containing proteins, including Grp1 signaling partner (GRSP)1/mKIAA1013 (47), Grp1-associated scaffold protein (GRASP)/tamalin (48,49), cytohesin-associated scaffold protein (CASP)/Cybr/cytohesininteracting protein (CYTIP) (50 -52), and interaction protein for cytohesin exchange factor (IPCEF) 1/KIAA0403 (53). Although CASP probably interacts specifically with cytohesin-1, GRSP1, GRASP, and IPCEF can bind to most cytohesin proteins.…”

Section: Journal Of Biological Chemistrymentioning

confidence: 99%

Cytohesin-2/ARNO, through Its Interaction with Focal Adhesion Adaptor Protein Paxillin, Regulates Preadipocyte Migration via the Downstream Activation of Arf6

Torii

Miyamoto

Sanbe

et al. 2010

The formation of primitive adipose tissue is the initial process in adipose tissue development followed by the migration of preadipocytes into adipocyte clusters. Comparatively little is known about the molecular mechanism controlling preadipocyte migration. Here, we show that cytohesin-2, the guaninenucleotide exchange factor for the Arf family GTP-binding proteins, regulates migration of mouse preadipocyte 3T3-L1 cells through Arf6. SecinH3, a specific inhibitor of the cytohesin family, markedly inhibits migration of 3T3-L1 cells. 3T3-L1 cells express cytohesin-2 and cytohesin-3, and knockdown of cytohesin-2 with its small interfering RNA effectively decreases cell migration. Cytohesin-2 preferentially acts upstream of Arf6 in this signaling pathway. Furthermore, we find that the focal adhesion protein paxillin forms a complex with cytohesin-2. Paxillin colocalizes with cytohesin-2 at the leading edges of migrating cells. This interaction is mediated by the LIM2 domain of paxillin and the isolated polybasic region of cytohesin-2. Importantly, migration is inhibited by expression of the constructs containing these regions. These results suggest that cytohesin-2, through a previously unexplored complex formation with paxillin, regulates preadipocyte migration and that paxillin plays a previously unknown role as a scaffold protein of Arf guanine-nucleotide exchange factor.

“…Tamalin (also termed GRP1-associated scaffold protein) is a scaffold protein that comprises multiple protein-interacting domains (24,25). It possesses a PDZ domain, a leucine-zipper region, a proline-rich region, and a carboxyl-terminal PDZ binding motif (24,25).…”

mentioning

confidence: 99%

“…It possesses a PDZ domain, a leucine-zipper region, a proline-rich region, and a carboxyl-terminal PDZ binding motif (24,25). The PDZ domain of tamalin interacts with the carboxyl termini of group 1 and group 2 metabotropic glutamate receptors (mGluRs) and GABA B2 receptor (24), whereas the leucine-zipper region binds to the coiled coil region of guanine nucleotide exchange factor cytohesins (24, 25).…”

mentioning

confidence: 99%

Tamalin Is a Scaffold Protein That Interacts with Multiple Neuronal Proteins in Distinct Modes of Protein-Protein Association

Kitano

Yamazaki

Kimura

et al. 2003

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/ X11␤/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.Multimolecular protein assembly through protein-protein interaction is important as a general mechanism for diverse cellular functions in neuronal and other cells (reviewed in Refs. 1-5). Molecular assembly of protein complexes is built around one or more central scaffold proteins that contain multiple domains for protein-protein interaction. The 95-kDa postsynaptic density protein (PSD-95) 1 /discs-large/ZO-1 (PDZ) domain is a key protein-binding domain comprised of ϳ90 amino acid residues and interacts with a PDZ binding motif with the consensus sequences S/TXV/I/L (X is any amino acid) (6, 7). In neurons, postsynaptic PDZ domain-containing scaffold proteins, PSD-95 and S-SCAM, interact with a number of membrane and cytoplasmic proteins, including NMDA receptors (7-9) and SAP90/PSD-95-associated proteins (SAPAPs) (also called guanylate kinase-associated proteins/human Discs Large-associated proteins) (10 -12). PSD-95 and S-SCAM are localized at the PSD of excitatory synapses and play an important role in functional assembly of a postsynaptic macromolecular complex. The PDZ domain-containing scaffold proteins are also important in subcellular trafficking of their partner proteins (reviewed in Refs. 1, 4, and 13). LIN-2, LIN-7, and LIN-10 of Caenorhabditis elegans are all PDZ domain-containing proteins and are important for the proper localization of the LET-23 receptor tyrosine kinase (13). LIN-2 is the homolog of mammalian scaffold protein CASK, which was simultaneously discovered as a protein interacting with the cell surface protein neurexin (14, 15). The mammalian homolo...