Identification of Splicing Variants of Rapostlin, a Novel Rnd2 Effector that Interacts with Neural Wiskott-Aldrich Syndrome Protein and Induces Neurite Branching

Abstract

Rho family GTPases regulate neuronal morphology.Rnd subfamily is a new branch of Rho family GTPases. Of these GTPases, Rnd2 is specifically expressed in brain. We recently identified Rapostlin as a novel effector of Rnd2. Rapostlin induces neurite branching in response to Rnd2 in PC12 cells. During the cloning of Rapostlin, we have found two mainly expressed splicing variants of Rapostlin (renamed as RapostlinL), RapostlinM and RapostlinS, lacking 29 residues and 61 residues within the unique insert region at the center, respectively, and three minor variants, RapostlinLd, RapostlinMd, and RapostlinSd, each with the identical five-amino acid deletion from RapostlinL, RapostlinM, and RapostlinS, respectively. RapostlinL is predominantly expressed in brain, whereas RapostlinS is expressed ubiquitously. In a dot-blot assay, all splicing variants bind to Rnd2 in a GTP-dependent manner. However, RapostlinM and RapostlinS induce less neurite branching when coexpressed with Rnd2 in PC12 cells, indicating that the insert region is important for the branching activity of RapostlinL. All splicing variants bind to N-WASP in vitro and in vivo through the SH3 domain at the carboxyl terminus, and the SH3 domain is essential for branching activity of RapostlinL. In immunoprecipitation experiments, Rnd2 reduces RapostlinL-N-WASP interaction, whereas it has little effect on the interaction of RapostlinM or RapostlinS with N-WASP. Therefore, we found that functionally different splicing variants of Rapostlin have different responses to Rnd2 in association with N-WASP.

Regulation of Neuronal Morphology by Toca-1, an F-BAR/EFC Protein That Induces Plasma Membrane Invagination

“…Immunoblotting-Immunoblotting was performed as described previously (11). Briefly, proteins were separated by SDS-10% PAGE and were electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA).…”

“…Plasmid Constructions and Antibodies-Rat Toca-1 was cloned as described previously (11) using RT-PCR with rat brain RNA as a template. cDNAs encoding the full-length Toca-1L (amino acids 2-609), Toca-1S (amino acids 2-551), Toca-1L-K576 (amino acids 2-609), Toca-1L-IST (amino acids 2-609), Toca-1L-⌬C (amino acids 2-371), Toca-1L-⌬N (amino acids 389 -609), and Toca-1L-QQ (amino acids 2-609) were subcloned into expression vector pCMV (Clontech, Mountain View, CA) encoding an initiating Met followed by the Myc epitope tag or enhanced green fluorescent protein (EGFP) sequence at the N terminus.…”

“…Therefore, we renamed Toca-1 as Toca-1S, and named the lon-ger variant as Toca-1L. We have previously reported that Rapostlin/formin-binding protein 17 (FBP17; hereafter referred to as Rapostlin), a Toca-1 paralog, also has two splicing variants with and without the insert region between the F-BAR/EFC domain and the HR1: RapostlinL and RapostlinS, respectively (11,17). The conserved splicing pattern suggests that the presence of the insert region may make a functional difference.…”

Direct Interaction of Rnd1 with FRS2β Regulates Rnd1-induced Down-regulation of RhoA Activity and Is Involved in Fibroblast Growth Factor-induced Neurite Outgrowth in PC12 Cells

“…To examine direct interaction between the FRS2␤ and Rnd1, dot blot assay was performed as described previously (21). Five g of GST and GST-fused FRS2␤ (amino acids 247-493) were spotted onto a nitrocellulose membrane and allowed to dry for 1 h at room temperature.…”

COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression

“…Accordingly, a previous study demonstrated that the interaction of Rnd2 with plexin D1 inhibits neurite outgrowth in cortical neurons (Uesugi et al, 2009). Finally, because Rnd2 modulates actin dynamics through interaction with the WASP proteins (Kakimoto et al, 2004), it is highly possible that increased Rnd2 levels might alter BSC neurite outgrowth, a process that is at the base of both glia-guided migration and CC formation.…”