Distinct subcellular localization of alternative splicing variants of EFA6D, a guanine nucleotide exchange factor for Arf6, in the mouse brain

Abstract

EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc.

Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions

“…Previous studies using primary cultured neurons implicated the EFA6-Arf6 pathway in neuronal processes related to the formation of neural circuits, such as the formation of dendrites [26, 27] and dendritic spines [9] and axonal transport [28]. We have previously shown that three members of the EFA6 family, EFA6A, EFA6C and EFA6D, are abundantly expressed in the adult mouse brain with distinct spatiotemporal patterns [17, 19, 20, 25, 26, 29]. However, the functional significance of individual EFA6 members is unknown at the whole animal level.…”

“…First, Arf6 can be activated by several Sec7-containing Arf GEFs: EFA6A–D in the EFA6 family [16–18, 20], BRAG1–3 in the BRAG family [34, 36, 44] and cytohesin 1–3 in the cytohesin family [45, 51]. Indeed, Purkinje cells abundantly express EFA6D [29], BRAG2 (our unpublished data), BRAG3 [36] and cytohesin 1–3 [51, 52] as well as EFA6C. Although the present immunoblot analyses failed to detect any compensatory changes in the protein expression of these Arf6 GEFs, it is still possible that their subcellular localization or GEF activities may be changed in Purkinje cells lacking EFA6C.…”

“…No. 17404-1-AP, ProteinTech, Rosemont, IL), EFA6D [29], BRAG1 [34], BRAG2 [35], BRAG3 [36], cytohesin-2 [37], Arf6 [38], calbindin (Cat. No.…”

“…The EFA6A-Arf6 pathway was shown to regulate dendritic formation [26, 27], maturation and maintenance of dendritic spines [9, 11], and directionality of axonal transport [28] in primary cultured neurons. EFA6D is widely expressed throughout the brain and is localized to various subcellular compartments in hippocampal neurons, including cell bodies, dendritic shafts and spines, axons and presynaptic terminals [20, 29]. A single nucleotide polymorphism in the human EFA6D gene was reportedly associated with alcohol drinking behaviors and neuronal activity in the prefrontal cortex during the go/no-go executive control task, suggesting modulatory roles of EFA6D in addictive and cognitive behaviors [30].…”

HGF-induced migration depends on the PI(3,4,5)P3-binding microexon-spliced variant of the Arf6 exchange factor cytohesin-1

“…In addition, there are multiple splice variants of Arf GEFs. Some differ by an entire domain, whereas others involve microexons (Ogasawara et al, 2000; Fukaya et al, 2016). The best-characterized microexon splice variants are two isoforms of cytohesin-2.…”