Identification of splicing variants of Rapostlin, a novel RND2 effector that interacts with neural Wiskott-Aldrich syndrome protein and induces neurite branching.
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.
Kakimoto
Tetsuhiro
T
Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Katoh
Hironori
H
Negishi
Manabu
M
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D016501:Neurites / Q000502:physiology*; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology
C470009:Fnbp1 protein, rat; D009419:Nerve Tissue Proteins; C495771:WASL protein, human; C495770:Wasl protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C470010:Rnd2 protein, rat; D020741:rho GTP-Binding Proteins