Abstract
Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.
Highlights
Small GTPase Rab17 regulates dendritic morphogenesis of hippocampal neurons, but its activation mechanism is completely unknown
Screening for Rab17-guanine nucleotide exchange factor (GEF) by Yeast Two-hybrid Assays with a GDP-locked Rab17 Mutant as Bait—To identify a Rab17-GEF molecule(s) that functions in neurons, we focused on previous reports showing that Rab-GEFs often physically interact with their substrate GDP-Rabs, e.g. DENND1/RME-4 interacts with Rab35 [14], ALS2 with Rab5 [23], Rin1 with Rab5 [35], Varp with Rab21 [36], and Rabin8 with Rab8 [7]
Several lines of evidence indicated that Rabex-5, but not ALS2, is likely to function as a Rab17-GEF in mouse hippocampal neurons: (i) overexpression of the GEF domain of Rabex-5, but not of ALS2, in hippocampal neurons promoted the dendritic localization of Rab17 (Figs. 3 and 4), (ii) knockdown of endogenous Rabex-5, but not of ALS2, in hippocampal neurons decreased the dendritic localization of Rab17 (Fig. 5, H and I), (iii) the total dendrite length of Rabex-5 knockdown neurons was partially restored by forced activation of Rab17, i.e. expression of Rab17-Q77L (Fig. 6F)
Summary
Small GTPase Rab regulates dendritic morphogenesis of hippocampal neurons, but its activation mechanism is completely unknown. Small GTPase Rab has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab had been identified. In this study we screened for Rab17-GEFs by using a GDP-locked Rab mutant as bait and identified Rabex-5 [22] and ALS2 (amyotrophic lateral sclerosis 2) [23], both of which were originally described as Rab5-GEFs, as putative Rab17-GEFs. We found that Rabex-5, but not ALS2, is required for stage-dependent movement of Rab protein from the cell body to the dendrites of mouse hippocampal neurons. We discuss the possible functions of Rabex-5 in neurite morphogenesis of hippocampal neurons based on our findings
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