Recent advances in molecular biology have shown GTPases and phosphoproteins to be the paramount molecular switches utilized intracellularly in biological systems. The origins of the GTPase switch appear to be almost as ancient as life itself, and through evolution nature has adapted this switch to a variety of purposes. In this two-volume work a broad survey of the major classes of GTPases is presented. The role of GTPases in ensuring accuracy during protein translation, a new look at the trimeric G-protein cycle, the molecular function of ARF in vesicle coating, the emerging role of the dynamin family in vesicle transfer, GTPases which activate GTPases during nascent protein translocation, and the many roles of ras-related proteins in growth, cytoskeletal polymerization, and vesicle transfer, are all described in 80 chapters by the leading authorities in their fields. Both detailed knowledge of specific systems or proteins and general principles of structure and function are offered. Much of this information has never been published before. At the rate the extended family of GTPases is growing it becomes increasingly unlikely that we will again get it to sit for a group portrait such as this. Therefore, the volume has the chance to become the reference work for GTPases.

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.

The nectin family of Ca 2+-independent immunoglobulin-like cell–cell adhesion molecules contains four members. Nectins, which have three Ig-like domains in their extracellular region, form cell–cell adherens junctions cooperatively with cadherins. The whole extracellular regions of nectin-1 (nectin-1-EC) and nectin-2 (nectin-2-EC) were expressed in Escherichia coli as inclusion bodies, solubilized in 8 M urea and then refolded by rapid dilution into refolding solution. The refolded proteins were subsequently purified by three chromato-graphic steps and crystallized using the hanging-drop vapour-diffusion method. The nectin-1-EC crystals belonged to space group P2 1 3 and the nectin-2-EC crystals belonged to space group P6 1 22 or P6 5 22.

The tumor suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumors. APC interacts with the Rac1- and Cdc42-specific guanine-nucleotide exchange factors (GEF), Asef and Asef2, which contain an APC-binding region (ABR) in addition to Dbl homology, Pleckstrin homology (PH) and Src homology 3 (SH3) domains. APC stimulates the GEF activity of Asef and Asef2, and thereby regulates cell adhesion and migration. Here we show that Asef2, but not Asef, interacts with Neurabin2/Spinophilin, a scaffold protein that binds to Filamentous actin (F-actin). In response to hepatocyte growth factor (HGF) treatment of HeLa cells, Asef2, Neurabin2 and APC were induced to accumulate and colocalize in lamellipodia and membrane ruffles. Neurabin2 did not affect the GEF activity of Asef2. RNA interference experiments showed that Asef2, Neurabin2 and APC are involved in HGF-induced cell migration. Furthermore, knockdown of Neurabin2 resulted in the suppression of Asef2-induced filopodia formation. These results suggest that Asef2, Neurabin2 and APC cooperatively regulate actin cytoskeletal organization and are required for HGF-induced cell migration.

This article addresses an evolutionary programming (EP) algorithm technique-based and proportional-integral-derivative (PID) control methods are established to guarantee synchronization of the master and slave Rikitake chaotic systems. For PID synchronous control, the evolutionary programming (EP) algorithm is used to find the optimal PID controller parameters kp, ki, kd by integrated absolute error (IAE) method for the convergence conditions. In order to verify the system performance, the basic electronic components containing operational amplifiers (OPAs), resistors, and capacitors are used to implement the proposed chaotic Rikitake systems. Finally, the experimental results validate the proposed Rikitake chaotic synchronization approach.

Cell adhesion molecules (CAMs) have been implicated in the control of a wide variety of cellular processes, such as cell adhesion, polarization, survival, movement, and proliferation. Nectins have emerged as immunoglobulin-like CAMs that participate in calcium-independent cell-cell adhesion by homophilic and heterophilic trans-interactions with nectins and nectin-like molecules. Nectin-based cell-cell adhesion exerts its function independently or in cooperation with other CAMs including cadherins and is essential for the formation of intercellular junctions, including adherens junctions, tight junctions, and puncta adherentia junctions. Nectins cis-interact with integrin alpha(v)beta(3) and platelet-derived growth factor receptor and facilitate their signals to regulate the formation and integrity of intercellular junctions and cell survival. Nectins intracellularly associate with peripheral membrane proteins, including afadin and Par-3. This review focuses on recent progress in understanding the interactions of nectins with other transmembrane and peripheral membrane proteins to exert pleiotropic functions.

Necl‐5 is an immunoglobulin‐like molecule that was originally identified as a poliovirus receptor. Although Necl‐5 expression is often up‐regulated in cancer cells, its pathophysiological significance in the development of cancer remains unclear. We investigated the roles of Necl‐5 in the development of colitis‐associated neoplasia. Necl‐5‐deficient mice were generated and treated with dimethylhydrazine (DMH) and/or dextran sodium sulphate (DSS) to induce colitis and its associated neoplasias. Colon tissues were examined for histology, Ki‐67 expression by immunohistochemistry and K‐ras gene mutation. Colon tumours occurred significantly less frequently in heterozygous (Necl‐5+/−) or homozygous Necl‐5‐deficient (Necl‐5−/−) mice than in wild‐type (WT) mice with DMH/DSS treatment. Total ulcer index and inflammatory cell infiltration were significantly lower in Necl‐5−/− mice than in WT mice with DSS alone or DMH/DSS treatment. Colon tumours in both WT and Necl‐5−/− mice showed high cell proliferation ability but lacked K‐ras mutation. The total Ki‐67 labelling index in non‐neoplastic colon epithelium was significantly higher in WT (45.9 ± 0.94) than in Necl‐5+/− (34.3 ± 1.40) or Necl‐5−/− (27.7 ± 1.15) mice with DMH/DSS treatment (p < 0.001). Necl‐5 plays a role in the development of colitis‐associated cancer by up‐regulating colonic mucosal cell proliferation. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cell adhesion molecules (CAMs) have been implicated in the control of a wide variety of cellular processes, such as cell adhesion, polarization, survival, movement, and proliferation. Nectins have emerged as immunoglobulin-like CAMs that participate in calcium-independent cell-cell adhesion by homophilic and heterophilic trans-interactions with nectins and nectin-like molecules. Nectin-based cell-cell adhesion exerts its function independently or in cooperation with other CAMs including cadherins and is essential for the formation of intercellular junctions, including adherens junctions, tight junctions, and puncta adherentia junctions. Nectins cis-interact with integrin αvβ3 and platelet-derived growth factor receptor and facilitate their signals to regulate the formation and integrity of intercellular junctions and cell survival. Nectins intracellularly associate with peripheral membrane proteins, including afadin and Par-3. This review focuses on recent progress in understanding the interactions of nectins with other transmembrane and peripheral membrane proteins to exert pleiotropic functions.

In response to chemoattractants, migrating cells form protrusions, such as lamellipodia and filopodia, and structures, such as ruffles over lamellipodia, focal complexes and focal adhesions at leading edges. The formation of these leading edge structures is essential for directional cell movement. Nectin-like molecule-5 (Necl-5) interacts in cis with PDGF receptor and integrin alpha(v)beta(3), and enhances the activation of signalling molecules associated with these transmembrane proteins, which results in the formation of leading edge structures and enhancement of directional cell movement. When migrating cells come into contact with each other, cell-cell adhesion is initiated, resulting in reduced cell velocity. Necl-5 first interacts in trans with nectin-3. This interaction is transient and induces down-regulation of Necl-5 expression at the cell surface. resulting in reduced cell movement. Cell proliferation is also suppressed by the down-regulation of Necl-5, because the inhibitory effect of Necl-5 on Sprouty 2, a negative regulator of the Ras signalling, is diminished. PDGF receptor and integrin alpha(v)beta(3), which have interacted with Necl-5, then form a complex with nectin, which initiates cell-cell adhesion and recruits cadherin to the nectin-based cell-cell adhesion sites to form stable adherens junctions. The formation of adherens junctions stops cell movement, in part through inactivation of integrin alpha(v)beta(3) caused by the transinteraction of nectin. Thus. nectin and Necl-5 play key roles in the regulation of cell movement and proliferation.

Necl-5 is an immunoglobulin (Ig)-like molecule that was originally identified as a poliovirus receptor and is often upregulated in cancer cells. We recently found that it colocalizes with integrin alpha(v)beta(3) at the leading edges of moving cells and enhances growth factor-induced cell movement and proliferation. Upon cell-cell contact, Necl-5 is removed from the cell surface by its trans-interaction with the cell adhesion molecule nectin-3, resulting in reduced cell movement and proliferation. Here, we investigated the role of Necl-5 in the interaction of cancer cells with platelets. Necl-5 was upregulated in CT26 cells, a colon adenocarcinoma cell line. When CT26 cells were injected into the tail vein of mice, they were arrested in the pulmonary vessels by adhering to platelets and subsequently metastasized to the lungs. Overexpression of Necl-5 in CT26 cells enhanced this metastasis, while inhibition of the trans-interaction of Necl-5 with CD226 by an anti-Necl-5 monoclonal antibody reduced the metastasis. Depletion of platelets by treatment with a rabbit anti-mouse platelet serum reduced the Necl-5-enhanced metastasis in mice. Thus, the trans-interaction of upregulated Necl-5 in cancer cells with its counter-receptor in platelets, probably CD226, is critical for efficient metastasis of cancer cells to the lungs.

Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology.

Nectin is an immunoglobulin-like cell-cell adhesion molecule and forms adherens junctions cooperatively with cadherin. Trans-interaction of nectin induces activation of Rap1, Cdc42, and Rac small G proteins. The activity of these small G proteins can be analyzed by the pull-down assay using GST-Ral-GDS fusion protein for Rap1 and GST-PAK-CRIB for Cdc42 and Rac. The fluorescent resonance energy transfer (FRET) system is also available to spatially and temporally detect the activity of these small G proteins in the living cells. In addition to these assays, the activity of Cdc42 and Rac is indirectly, but easily, evaluated by the cell-spreading assay to examine formation of filopodia and lamellipodia, respectively. To clearly explore the effect of trans-interacting nectin on the small G proteins, we use L fibroblasts stably expressing nectin-1 (nectin-1-L cells) and the extracellular domain of nectin-3 fused to human IgG Fc (Nef-3). Treatment of nectin-1-L cells with Nef-3 remarkably increases both the amount of the GTP-bound form and the FRET efficiency of all Rap1, Cdc42, and Rac small G proteins. In the cell-spreading assay, Cdc42 and Rac activated in this way promote the formation of filopodia and lamellipodia, respectively. Here, we focus on how the activity of Cdc42 and Rac induced by trans-interacting nectin is examined by use of the pull-down and the cell-spreading assays.

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of an extracellular signal, such as hepatocyte growth factor (HGF)/scatter factor. Rab5 small G protein has been implicated in the HGF-induced endocytosis of E-cadherin, but the molecular mechanism for the regulation of Rab5 activity remains unknown. We first studied this mechanism by using the cell-free assay system for the endocytosis of E-cadherin of the AJ-enriched fraction from rat livers. HGF induced activation of Ras small G protein, which then bound to RIN2, a Rab5 GDP/GTP exchange factor with the Vps9p-like guanine nucleotide exchange factor and Ras association domains, and activated it. Activated RIN2 then activated Rab5, eventually inducing the endocytosis of E-cadherin. We then studied whether RIN2 was involved in the HGF-induced endocytosis of E-cadherin in intact Madin-Darby canine kidney cells. RIN2 localized at the cell-cell adhesion sites, and its guanine nucleotide exchange factor activity was required for the HGF-induced endocytosis of E-cadherin in Madin-Darby canine kidney cells. These results indicate that RIN2 connects Ras to Rab5 in the HGF-induced endocytosis of E-cadherin.

CAST is a novel cytomatrix at the active zone (CAZ)‐associated protein. In conventional brain synapses, CAST forms a large molecular complex with other CAZ proteins, including RIM, Munc13‐1, Bassoon, and Piccolo. Here we investigated the distribution of CAST and its structurally related protein, ELKS, in mouse retina. Immunofluorescence analyses revealed that CAST and ELKS showed punctate signals in the outer and inner plexiform layers of the retina that were well‐colocalized with those of Bassoon and RIM. Both proteins were found presynaptically at glutamatergic ribbon synapses, and at conventional GABAergic and glycinergic synapses. Moreover, immunoelectron microscopy revealed that CAST, like Bassoon and RIM, localized at the base of synaptic ribbons, whereas ELKS localized around the ribbons. Both proteins also localized in the vicinity of the presynaptic plasma membrane of conventional synapses in the retina. These results indicated that CAST and ELKS were novel components of the presynaptic apparatus of mouse retina. J. Comp. Neurol. 495:480–496, 2006. © 2006 Wiley‐Liss, Inc.

Adherens junctions (AJs) and tight junctions (TJs) comprise a junctional complex which plays key roles not only in cell adhesion and polarization but also in regulation of cell movement and proliferation in epithelial cells. E-Cadherin and nectin are major cell–cell adhesion molecules (CAMs) at AJs, whereas claudin is a major CAM at TJs. We have shown that the cadherin-based cell–cell adhesion is not formed in MDCK cells in which annexin II, a Ca 2+ - and phospholipid-binding protein, is knocked down. Here, we found that TJs and the nectin-based cell–cell adhesions were formed in annexin II-knockdown cells. The formation of TJs in annexin II-knockdown MDCK cells required the nectin-based cell–cell adhesion and afadin, a nectin- and actin-filament-binding protein. In addition, it required the activation of Cdc42 and Rac small G proteins and subsequent reorganization of the IQGAP1-dependent actin cytoskeleton which were induced by the nectin-based cell–cell adhesion. These results indicate that the nectin-based cell–cell adhesion and afadin, but not the cadherin-based cell–cell adhesion, are necessary for the formation of TJs and that the signaling by nectin and the subsequent reorganization of the actin cytoskeleton are also necessary for the formation of TJs under certain conditions.

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.