• Molecules and Cells
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• v.40 no.7
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• pp.495-502
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• 2017

The $5-HT_6R$ has been considered as an attractive therapeutic target in the brain due to its exclusive expression in the brain. However, the mechanistic linkage between $5-HT_6Rs$ and brain functions remains poorly understood. Here, we examined the effects of $5-HT_6R$-mediated cell morphological changes using immunocytochemistry, Western blot, and live-cell imaging assays. Our results showed that the activation of $5-HT_6Rs$ caused morphological changes and increased cell surface area in HEK293 cells expressing $5-HT_6Rs$. Treatment with 5-HT specifically increased RhoA-GTP activity without affecting other Rho family proteins, such as Rac1 and Cdc42. Furthermore, live-cell imaging in hippocampal neurons revealed that activation of $5-HT_6Rs$ using a selective agonist, ST1936, increased the density and size of dendritic protrusions along with the activation of RhoA-GTP activity and that both effects were blocked by pretreatment with a selective $5-HT_6R$ antagonist, SB258585. Taken together, our results show that $5-HT_6R$ plays an important role in the regulation of cell morphology via a RhoA-dependent pathway in mammalian cell lines and primary neurons.

• Journal of Plant Biology
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• v.33 no.3
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• pp.211-215
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• 1990

There is a family of homologous proteins known to small GTP-inding proteins which have a GTP binding domains and GTPase activity with molecular weight of about 20000 in mammalian tissues. Recently at least 20 different small GTP-binding proteins including three rasproto-oncogene, smg25, rho, and ral gene products were identified. These proteins play a central role in cellular prolifration, neoplasia, signal transduction, terminal differentiation, and secretory process of the cells. In this review, I have briefly compiled current information on the different areas of research in the small GTP-binding proteins in an attempt to convey an overall view of the fundamental role that this family of protein in normal cellular processes. Moreover, furture goals of research in the small GTP-binding proteins as well as the possible existence of this family of proteins in plant cells were discussed.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.192-197
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• 2007

Two distinct morphological features, leading edge and uropod, in mobile T lymphocyte are crucial for efficient directional movement. The uropod is a unique rear protrusion in migrating lymphocytes, in which several proteins, including CD44, ERM (ezrin/radixin/moesin), and F-actin cytoskeleton are concentrated and concerted. F-actin cytoskeleton is a basic mold for the shape maintenance. Rho A small GTPase acts as cytoskeleton organizer, So far, various pathways potentially can induce the Rho activation. PDZ domain is able to increase active Rho A form (Rho-GTP) level, reorganize F-actin cytoskeleton, disrupts the uropod structure and cell migration was diminished, suggesting that signaling pathways between Rho and F-artin cytoskeleton are related to uropod formation.

• Journal of Life Science
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• v.29 no.2
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• pp.256-264
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• 2019

Lymphotoxin ${\beta}$ receptor ($LT{\beta}R$), a member of the tumor necrosis factor receptor family, plays an important role in lymphoid tissue's architecture and organogenesis. In contrast, MLCK and ROCK play critical roles in the regulation of stress fiber (SF) formation in cells. To determine whether $LT{\beta}R$ stimulation in fibroblastic reticular cells (FRCs) is involved in these signaling pathways, myosin light chain kinase inhibitor-7 (ML-7) was used to inhibit them. ML7-treated FRCs completely blocked SFs and showed retraction and shrinkage processes comparable to those observed in agonistic anti-$LT{\beta}R$ antibody-treated cells. The inhibition of ROCK activity with Y27632-induced changes in actin cytoskeleton organization and cell morphology in FRCs. Actin bundles rearranged into SFs, and phospho-myosin light chain (p-MLC) co-localized in FRCs. We checked the level of Rho-guanosine diphosphate (RhoGDP)/guanosine triphosphate (GTP) exchange activity using FRC lysate. When $LT{\beta}R$ was stimulated with agonistic anti-$LT{\beta}R$ antibodies, Rho-GDP/GTP exchange activity was markedly reduced. Regarding $LT{\beta}R$ signaling with a focus on MLCK inhibition, we showed that the phosphorylation of MLCs was reduced by $LT{\beta}R$ stimulation in FRCs. Cytoskeleton components, such as tubulin, b-actin, and phospho-ezrin proteins acting as membrane-cytoskeleton linkers, were produced in de-phosphorylation, and they reduced expression in agonistic anti-$LT{\beta}R$ antibody-treated FRCs. Collectively, the results suggested that MLCK and ROCK were simultaneously responsible for SF regulation triggered by $LT{\beta}R$ signaling in FRCs.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.3
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• pp.175-182
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• 2022

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

• BMB Reports
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• v.30 no.5
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• pp.337-341
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• 1997

In order to investigate the role of a small GTP-binding protein RhoA in lysophosphatidic acid (LPA)-induced stress fiber formation, C3 ADP-ribosyltransferase was prepared by expressing in E. coli and then applied to Rat-2 fibroblasts. C3 transferase isolated from E. coli was as effective as the toxin from Clostridium botulinum in ADP-ribosylation of RhoA. Incubation of the cells with C3 transferase for 2 days induced ADP-ribosylation of RhoA by a dose-dependent manner, with a sub-maximal induction at $25\;{\mu}g/ml$. As expected, LPA-induced stress fiber formation was completely blocked by pre-incubation with C3 transferase for 2 days. However, exogenously added C3 transferase had no significant effect on the formation of phosphatidylethanol by LPA. These results suggested that phospholipase D was not activated by RhoA in the LPA-induced stress fiber formation.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.51-57
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• 2010

It was hypothesized that NaF induces calcium sensitization in $Ca^{2+}$-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with $\beta$-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to $Ca^{2+}$ (decreased $EC_{50}$ and increased $E_{max}$). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in $Ca^{2+}$-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and $GTP{\gamma}S$-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a $Ca^{2+}$ dependent manner in $\beta$-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.

• Molecules and Cells
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• v.27 no.2
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• pp.167-173
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• 2009

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for $Ca^{2+}$-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by $GTP{\gamma}S$ was not desensitized. TPRC4 activation by $GTP{\gamma}S$ was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with $pK_a$ of 7.3. Finally, TPRC4 activation by $GTP{\gamma}S$ was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles.

• Journal of Life Science
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• v.27 no.10
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• pp.1199-1206
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• 2017

The lymphotoxin ${\beta}$ receptor ($LT{\beta}R$), a member of the tumor necrosis factor receptor family, plays an important role in lymphoid tissue's architecture and organogenesis. We found that $LT{\beta}R$ stimulation induced changes in stress fibers (SFs) in fibroblastic reticular cells (FRCs). MLCK and ROCK play critical roles in the regulation of SF formation in cells. The present study was performed to investigate the antifibrotic effects on SF regulation of $LT{\beta}R$ signaling, with a focus on MLCK inhibition. The effect of $LT{\beta}R$ on the SF change was analyzed using immunoblot and fluorescence assays and agonistic $anti-LT{\beta}R$ antibody-treated FRCs. In addition, we checked the level of Rho-guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange activity with FRC lysate. Phospho-ezrin proteins acting as membrane-cytoskeleton linkers completely de-phosphorylated in agonistic $anti-LT{\beta}R$ antibody-treated FRCs. The actin bundles rearranged into SFs, where phospho-myosin light chain (p-MLC) co-localized in FRCs. ML7-treated FRCs completely blocked SFs and showed retraction and shrinkage processes comparable to those observed in agonistic $anti-LT{\beta}R$ antibody-treated cells. Inhibition of ROCK activity induced changes in the actin cytoskeleton organization; however, some SFs remained in the cells, while they were completely disrupted by MLCK inhibition with ML7. We showed that the phosphorylation of MLC was completely abolished with $LT{\beta}R$ stimulation in FRCs. When $LT{\beta}R$ was stimulated with the agonistic $anti-LT{\beta}R$ antibody, the Rho-GDP/GTP exchange activity was reduced, however, the activity was not completely abolished. Collectively, the results illustrated that MLCK was potently responsible for the SF regulation triggered via $LT{\beta}R$ signaling in FRCs.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.222.1-222.1
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• 2003

The signal transduction pathway leading to the activation of the p70s6k plays an important role in the progression of cells from G0/G1 to S phase of the cell cycle but remains incompletely characterized. We investigated the role of the Rho family G protein Rac1 in H2O2-mediated p70s6k activation. Transient expression of a dominant negative mutants of the small GTP-binding proteins Rac1 (Rac1N17) and Cdc42(Cdc42N17) showed reduced levels of slower migration on Western blots of one-dimensional SDS-PAGE in p70s6k and ERK1/2 by PDFG stimulation. (omitted)