Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells

ABSTRACT The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.

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Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells

The Journal of Cell Biology ◽

10.1083/jcb.200308070 ◽

2003 ◽

Vol 163 (5) ◽

pp. 1067-1076 ◽

Cited By ~ 29

Author(s):

Mitsunobu Hoshino ◽

Shun Nakamura

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Small Gtpase ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Binding Motif ◽

Mutant Proteins ◽

Neuronal Signaling ◽

Voltage Dependent ◽

Mitogen Activated Protein

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal–binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin–CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.

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Rab20, a novel Rab small GTPase that negatively regulates neurite outgrowth of PC12 cells

Neuroscience Letters ◽

10.1016/j.neulet.2017.10.056 ◽

2018 ◽

Vol 662 ◽

pp. 324-330 ◽

Cited By ~ 5

Author(s):

Mai E. Oguchi ◽

Kan Etoh ◽

Mitsunori Fukuda

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Small Gtpase

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Rab13 Regulates Neurite Outgrowth in PC12 Cells through Its Effector Protein, JRAB/MICAL-L2

Molecular and Cellular Biology ◽

10.1128/mcb.01067-09 ◽

2009 ◽

Vol 30 (4) ◽

pp. 1077-1087 ◽

Cited By ~ 56

Author(s):

Ayuko Sakane ◽

Kazufumi Honda ◽

Takuya Sasaki

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Cell Body ◽

Intramolecular Interaction ◽

Binding Protein ◽

Active Form ◽

Coiled Coil ◽

Actin Binding ◽

Effector Protein ◽

Lim Domains

ABSTRACT Neurite outgrowth is the first step in the processes of neuronal differentiation and regeneration and leads to synaptic polarization and plasticity. Rab13 small G protein shows an increased mRNA expression level during neuronal regeneration; it is therefore thought to be involved in this process. We previously identified JRAB (junctional Rab13-binding protein)/MICAL-L2 (molecules interacting with CasL-like 2) as a novel Rab13 effector protein. Here, we show that Rab13 regulates neurite outgrowth in the rat pheochromocytoma cell line PC12 through an interaction with JRAB/MICAL-L2. The expression of JRAB/MICAL-L2 alone inhibits neurite outgrowth, whereas coexpression of the dominant active form of Rab13 rescues this effect. We also demonstrate an intramolecular interaction between the N-terminal calponin-homology (CH) and LIM domains of JRAB/MICAL-L2 and the C-terminal coiled-coil domain. Finally, we show that the binding of Rab13 to JRAB/MICAL-L2 stimulates the interaction of JRAB/MICAL-L2 with actinin-4, an actin-binding protein, which localizes to the cell body and the tips of the neurites in PC12 cells. These results suggest that Rab13 and JRAB/MICAL-L2 may act to transfer actinin-4 from the cell body to the tips of neurites, where actinin-4 is involved in the reorganization of the actin cytoskeleton which results in neurite outgrowth.

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Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0277 ◽

2011 ◽

Vol 22 (20) ◽

pp. 3853-3860 ◽

Cited By ~ 24

Author(s):

Liang Wang ◽

Zhimin Liang ◽

Guangpu Li

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Critical Role ◽

Inhibitory Effect ◽

Small Gtpase ◽

Rab Gtpase ◽

Early Endosomes ◽

Ngf Signaling

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22–Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22–Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.

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