PDZ-scaffold protein, Tamalin promotes dendritic outgrowth and arborization in rat hippocampal neuron

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Abstract

Tamalin is a scaffold protein known to regulate membrane trafficking through its interaction with cytohesin-2/ARNO, guanine nucleotide exchange factor (GEF) on ADP-ribosylation factor (Arf) 1/6, and induces actin reorganization. However, the neuronal function of Tamalin is not well understood. Here, we report that Tamalin participates in neurite development through the association with exchange factor for Arf6 (EFA6A)/Arf6 signaling. In immature hippocampal neuron, Tamalin knockdown markedly reduced the dendritic outgrowth, the number of dendritic tips and the levels of filamentous actin (F-actin) and microtubule-associated protein 2 (MAP2) in dendrites. In addition, Tamalin colocalized with EFA6A and Arf6 in the dendritic shaft. Tamalin knockdown reduced the number, size, and intensity of endogenous EFA6A cluster, whereas overexpression of Tamalin showed opposite effects compared with those of knockdown. These results suggest that Tamalin is responsible for neuronal dendritic development via regulation of EFA6A/Arf6-mediated cytoskeleton dynamics.

Highlights

► Tamalin regulates dendritic outgrowth and branching in dendritic development. ► Tamalin forms a complex with EFA6A and Arf6 in dendrites. ► F-actin and MAP2 regulate dendritic development.

Introduction

Tamalin (also termed GRP1-associated scaffold protein, GRASP) is a scaffold protein that contains multiple protein-interacting domains, including alanine-rich, PDZ (PSD-95/discs-large/ZO-1), leucine-zipper, proline-rich domains and C-terminal PDZ binding motif [1], [2]. The PDZ domain of Tamalin binds to C-terminal PDZ binding motifs of metabotrophic glutamate receptors (mGluRs), gamma-aminobutyric acid B receptor 2 (GABAB2), SAPAP3, and Tamalin itself, whereas the leucine-zipper region interacts with the coiled coil domain of cytohesin-2/ARNO. In addition, Tamalin forms a direct complex with PDZ domains of S-SCAM, Mint2, and PSD-95 via its C-terminal PDZ binding motif [3].

In the rat brain membrane fractions, Tamalin forms multimolecular protein complex comprising not only mGluR1 but also c-Src, Fyn, and a protein phosphatase, SHP-2, suggesting that Tamalin is a novel signaling molecule mediating Syk signaling [4]. In addition, neurotrophin-3 (NT3) binding to TrkCT-1 causes the recruitment of Tamalin and cytohesin-2/ARNO to the TrkCT-1 cytoplasmic domain, which in turn produces the active Arf6-GTP form. Arf6-GTP activates Rac1 GTPase and induces membrane ruffling and the formation of cellular protrusions via actin reorganization [5]. Knockdown of Tamalin in Madin-Darby Canine Kidney (MDCK) cells prevents the association of ARNO and Dock180, and blocks ARNO-mediated Rac activation [6]. Especially, PSD-95, Tamalin-interacting synaptic scaffold protein, regulates negatively dendritic outgrowth and branching in an activity-dependent manner. In addition, PSD-95-mediated inhibition of dendritic branching is antagonized by cypin [7] which binds directly to tubulin heterodimers and promotes microtubule polymerization [8]. Recent study showed that Tamalin deficiency blocks electroconvulsive shock (ECS)-induced proliferation and neurite development of adult hippocampal progenitors in dentate gyrus [9].

Here, we report the involvement of Tamalin and EFA6A in neuronal dendritic development. Results from immunocytochemistry, overexpression, and knockdown suggest that cellular function of Tamalin in early neuronal development is associated with EFA6A/Arf6 pathway.

Section snippets

Expression and short hairpin RNA constructs

Rat Tamalin (full length, GenBank Accession No. AF374272.1) was subcloned into pRK5-myc (Clontech). For short hairpin RNA (shRNA) knockdown construct, oligonucleotide targeting the following region of Tamalin was subcloned into pSUPER.gfp/neo (Oligoengine); nt 1320–1338 of rat Tamalin, 5′-GTCCCAGCACAAAGAAGAA-3′.

Primary neuron culture, transfection, and immunocytochemistry

All experiments were approved by Korea University Institutional Animal Care & Use Committee. Cultured hippocampal neurons were prepared from embryonic (E18) Sprague Dawley rats (of

Expression patterns of Tamalin

Because Tamalin is a functionally novel protein, we investigated its expression pattern in the adult rat brain (6 weeks), using immunoblot analysis. Tamalin protein is widely expressed in various rat brain regions, including cortex, cerebellum, hippocampus, and olfactory bulb (Fig. 1A, upper panel). Expression levels of Tamalin protein gradually increased during postnatal rat brain development, similar to PSD-95 (Fig. 1A, bottom panel), but decreased in adult stage. We determined the subcellular

Discussion

Our results presented herein indicate that Tamalin promotes dendritic outgrowth and arborization via regulation of EFA6A/Arf6 pathway. Endogenous Tamalin is highly expressed during postnatal rat brain development. Knockdown of Tamalin suppressed dendritic outgrowth and reduced dendritic branching tips. In contrast, its overexpression increased the outgrowth and branching of dendrites. A notable feature of Tamalin is that it colocalizes with EFA6A via direct interaction in dendrites of

Acknowledgments

This work was supported by the Korea Science and Engineering Foundation (KOSEF) Grants 2011-0019227, 2011-0019229, and 2010K000830. We thank Dr. Eunjoon Kim (Korea Advanced Institute Science and Technology) for the kind gift of Arf6 and EFA6A antibodies.

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