Elsevier

Neuroscience Research

Volume 60, Issue 2, February 2008, Pages 199-212
Neuroscience Research

IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for Arf6 that interacts with PSD-95 at postsynaptic density of excitatory synapses

https://doi.org/10.1016/j.neures.2007.10.013Get rights and content

Abstract

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-d-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAa receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6.

Introduction

Excitatory synapses are mostly formed on bulbous heads of dendritic spines protruding from dendritic shafts. On the cytoplasmic face of the excitatory postsynaptic membrane lies an electron-dense structure called the postsynaptic density (PSD). The PSD is a highly organized laminar structure of the excitatory postsynaptic membrane, at which various glutamate receptors are densely accumulated. Glutamate receptors and their downstream signaling molecules are recruited to the PSD of excitatory synapses through interaction with multivalent scaffold proteins, such as PSD-95, ProSAP/Shank family, and Homer (Scannevin and Huganir, 2000, Kim and Sheng, 2004, Boeckers, 2006). Recent technical advances in proteomics have uncovered a variety of molecular components in the PSD (Walikonis et al., 2000, Jordan et al., 2004, Peng et al., 2004, Yoshimura et al., 2004). Among them, several regulators and effectors of small GTPases, including Ras, Rho, and Rap, are found to be highly concentrated at the PSD and mediate dynamic structural changes of dendritic spines through the organization of actin cytoskeleton (Lippman and Dunaevsky, 2005, Newey et al., 2005, Calabrese et al., 2006).

ADP ribosylation factors (Arfs) represent the Ras-related small GTPases and consist of six structurally related isoforms (Arf1–6). Among them, Arf6 is unique in its localization at the plasma membrane and endosome and its regulation of actin cytoskeleton as well as endosomal trafficking (Donaldson, 2003, D'Souza-Schorey and Chavrier, 2006). Arf6 has been shown to regulate the formation and maintenance of dendritic spines (Choi et al., 2006), the branching of axons and dendrites (Hernandez-Deviez et al., 2002, Hernandez-Deviez et al., 2004), exocytosis and endocytosis of synaptic vesicles (Vitale et al., 2002, Krauss et al., 2003) and receptor internalization (Krauss et al., 2003, Houndolo et al., 2005).

The activation of Arfs requires guanine nucleotide exchange factors (GEFs) that facilitate the exchange of GDP for GTP. Among the six groups of mammalian Arf-GEFs (Cox et al., 2004), those preferably activating Arf6, Arf6-GEFs, include ARNO/cytohesin-2 and GRP1/cytohesin-3 in the cytohesin family, four members (EFA6A-D) of the EFA6 (exchage factor for Arf6) family and ARF-GEP100 in the BRAG (brefeldin A-resistant Arf-GEF) family (Frank et al., 1998, Franco et al., 1999, Langille et al., 1999, Someya et al., 2001, Matsuya et al., 2005, Sakagami et al., 2006). To pursue how and where Arf6 activation is controlled by Arf6-GEFs, we and others explored their localization and functions, and have presented the evidence for their functional importance at the PSD of dendritic spines (Kitano et al., 2002, Matsuya et al., 2005, Choi et al., 2006, Sakagami et al., 2006).

SynArfGEF(Po) has recently been identified as a novel potential Arf-GEF that belongs to the BRAG family by screening of transcripts associated with the PSD (Inaba et al., 2004). In this study, we characterized the GEF activity for Arf6, cellular and subcellular localization of a Arf-GEF, IQ-ArfGEF/BRAG1, which was identified by the homology search with synArfGEF(Po) as a bait (Sakagami and Kondo, 2006). Furthermore, we found that it can directly bind to PSD-95 and forms a postsynaptic protein complex with NMDA glutamate receptors. A part of this study has preliminary been reported in the 19th Annual Meeting of the Japanese Neuroscience Society (Sakagami and Kondo, 2006).

Section snippets

Animals

C57BL/6 mice, guinea pigs and New Zealand white rabbits used in this study were carefully handled in accordance with the guidelines regarding the Care and Use of Laboratory Animals of Tohoku University Graduate School of Medicine.

cDNA cloning

The mouse brain cDNA libraries were screened with a mixture of radiolabeled cDNA fragments encoding the Sec7 domains of rat synArfGEF(Po) and mouse KIAA0522 at a low stringent condition. Several overlapping cDNA clones were isolated and the nucleotide sequences were

Structure of mouse IQ-ArfGEF/BRAG1

By using the sequence of the Sec7 domain of synArfGEF(Po) as a bait, BLAST database search identified a mouse cDNA termed KIAA0522. KIAA0522 exhibited high homology (77.6%) with synArfGEF(Po), encoded a partial 642-amino acid protein and contained the Sec7 and PH domains, both of which were shared in the Arf-GEF family. To determine the complete primary structure, mouse brain cDNA libraries were further screened with a mixture of radioactive cDNA fragments corresponding to the Sec7 domains of

Discussion

Accumulative evidence highlights the existence of multiple GEFs and GAPs for small GTPases in the PSD and their functional involvement in the signal transduction at the downstream of NMDA receptors (Lippman and Dunaevsky, 2005, Newey et al., 2005, Calabrese et al., 2006). This study demonstrated that IQ-ArfGEF/BRAG1 is a GEF for Arf6 that forms a complex with NMDA receptors through the interaction with PSD-95 family proteins and exclusively localized at postysynaptic sites of excitatory

Acknowledgements

We greatly appreciate Dr. Hiroshi Takeshima (Kyoto University Graduate School of Pharmaceutical Sciences) for a mouse cDNA library, Dr. Kazuhisa Nakayama (Kyoto University Graduate School of Pharmaceutical Sciences) for the expression vectors for ARFs and GGA1-GAT-GST fusion protein, Dr. Jun-Ichi Miyazaki (Osaka University Graduate School of Medicine) for pCAGGS vector, and Dr. Sachiko Saino-Saito (Tohoku University Graduate School of Medicine) for her assistance of non-radioactive in situ

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