Research ReportDistinct spatiotemporal expression of EFA6D, a guanine nucleotide exchange factor for ARF6, among the EFA6 family in mouse brain
Introduction
ADP ribosylation factors (ARFs) of small GTPase are molecular switches that regulate various membrane dynamics. The mammalian ARFs comprise of three classes based on their sequence similarity. Class I ARFs including ARF1, 2, and 3 are involved in vesicle formation and transport between endoplasmic reticulum and Golgi complex. Little is known about the functions of class II ARFs including ARF4 and 5. In contrast, Class III ARF, ARF6, has recently been highlighted because of its unique functions in the crosstalk of plasma membranes and actin cytoskeleton dynamics and in the signaling of the endosomal pathway (Donaldson, 2003, Vitale et al., 2002).
Like other small GTPases, switching between active GTP-bound and inactive GDP-bound forms of ARF is tightly regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (Donaldson and Jackson, 2000, Jackson and Casanova, 2000, Jackson et al., 2000). GEFs activate ARFs by facilitating the exchange of the bound GDP for GTP, whereas GAPs inactivate ARFs by enhancing the intrinsic hydrolysis of the bound GTP to GDP. Increasing members of ARF GEFs has been identified to date, and all of them contain Sec7 domain, which is critical for the guanine exchange activity. A recent phylogenetic analysis has proposed that mammalian Sec7 domain-containing proteins can be classified into six families: cytohesin, BIG (brefeldin A-inhibited GEF)/Sec7, GBF (Golgi brefeldin A resistance factor)/GEA, BRAG (brefeldin A resistant ARF-GEF), EFA6 (exchange factor for ARF6), and FBX (F-box only protein 8, also called FBS (F-box/Sec7 protein)) (Cox et al., 2004).
The membrane trafficking and cytoskeleton reorganization are fundamental mechanisms that regulate neuronal differentiation including polarity establishment, migration, neurite extension, and synaptogenesis as well as mature neuronal functions such as neurotransmitter release and retrieval (Horton and Ehlers, 2003, Lecuit and Pilot, 2003, Sudhof, 2004). In line with this notion, ARF6 has been shown to regulate a variety of neuronal functions including the exocytosis and endocytosis of presynaptic vesicles, receptor internalization, and neurite formation (Claing et al., 2001, Galas et al., 1997, Hernandez-Deviez et al., 2002, Hernandez-Deviez et al., 2004, Miyazaki et al., 2005, Sheen et al., 2004).
Among the ARF GEFs that can activate ARF6, the EFA6 family has recently been shown to comprise of at least four isoforms (EFA6A–D) (Derrien et al., 2002, Franco et al., 1999). We have so far demonstrated that EFA6A and C exhibit distinct spatial expression patterns in adult mouse brain (Matsuya et al., 2005, Sakagami et al., 2004, Suzuki et al., 2002). Furthermore, the expression of a GEF-defective mutant of EFA6A has been shown to induce prominent dendritic formation in primary hippocampal neurons (Sakagami et al., 2004), suggesting the functional involvement of EFA6A-ARF6 pathway in the dendritic formation.
Because of the lack of information on the complete primary structure of EFA6D, no progress has been made in our understanding of the functional significance and diversity of the EFA6 family in brain. To address this issue, the present study determined the complete primary structure of mouse EFA6D and characterized its cellular and subcellular localization in comparison of that of other EFA6 isoforms.
Section snippets
Structure of mouse EFA6D
We screened a mouse brain oligo(dT)-primed cDNA library under low stringency conditions with a cDNA fragment of rat EFA6A encoding Sec7 domain and identified a 3201-bp cDNA clone (clone D5) that shared 47.2% homology with rat EFA6A. Because of the lack of a translation initiation codon, we further screened a mouse brain random-primed cDNA library with the 5′-region of clone D5 and obtained a 1905-bp cDNA clone (clone D101) that extended 1150 nucleotides from the 5′-end of clone D5 and contained
Discussion
In this study, we first determined the complete primary structure of mouse EFA6D and characterized its preferential GEF activity toward ARF6 by ARF pull-down assay. EFA6D contains Sec7 domain followed by PH domain and coiled coil domain in an amino-terminal order, which is consistent with the conserved structural features common to the EFA6 family. In contrast to the conserved carboxyl-terminal region, the amino-terminal regions are divergent among the EFA6 family. Unlike the other EFA6
Cloning and sequencing of mouse EFA6D
The cDNA fragment of rat EFA6A corresponding to nucleotides 835–1404 (Suzuki et al., 2002) amplified by PCR was used to screen brain cDNA libraries derived from adult C57BL/6J mice kindly provided by Dr. H. Takeshima (Tohoku University). The isolated cDNA clones were subjected to sequence analysis by an automatic DNA sequencer (Applied Biosystems, model 3100). The nucleotide sequence reported in this study has been submitted to the DDBJ/EMBL/GenBank™ with the accession number AB220685.
Construction of mammalian expression vector for EFA6D
To
Acknowledgments
The authors would like to thank Dr. J. Miyazaki (Osaka University Medical School) for kindly providing pCAGGS vector, Drs. Hye-Won Shin and Kazuhisa Nakayama (Kyoto University Graduate School of Pharmaceutical Sciences) for the expression vectors for ARFs and technical advice on ARF pull-down assay, and Dr. Hiroshi Takeshima (Tohoku University Graduate School of Medicine) for mouse cDNA libraries. This work is supported by a Grant-in-Aid for Scientific Research (C) (#17500219) to H.S. from the
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2018, Acta HistochemicaCitation Excerpt :It is thus possible that EFA6D is selectively involved in the Arf6-mediated membrane trafficking in these two steroidogenic cells. As described in Introduction, the present authors (HS and HK) have clarified the expression of this molecule in a wide variety of tissue cells in situ of mice in immunoblotting (Sakagami et al., 2006) and in several representative cells in immunohistochemistry, and this is the fourth study clarifying the localization of this molecule in mouse tissue cells. Regarding its expression in human testis, there has been a study showing an intense immunoreactivity in immunoblotting analysis (Kanamarlapudi, 2014).
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2014, Journal of Biological ChemistryCitation Excerpt :EFA6A, EFA6B, EFA6C, and EFA6D constitute EFA6 family members (37–39). Among them, EFA6A and EFA6C are predominantly expressed in the central nervous system, whereas EFA6B and EFA6D are widely distributed in various tissues (39, 40). EFA6A is abundantly expressed in hippocampal neurons and regulates dendrite formation (41).