title: Structure-function analyses of the small GTPase Rab35 and its effector protein centaurin-β2/ACAP2 during neurite outgrowth of PC12 cells.
abstract: The small GTPase Rab35 is a molecular switch for membrane trafficking that regulates a variety of cellular events. We previously showed that Rab35 promotes neurite outgrowth of nerve growth factor-stimulated PC12 cells through interaction with centaurin-β2 (also called ACAP2). Centaurin-β2 is the only Rab35-binding protein reported thus far that exclusively recognizes Rab35 and does not recognize any of the other 59 Rabs identified in mammals, but the molecular basis for the exclusive specificity of centaurin-β2 for Rab35 has remained completely unknown. In this study, we performed deletion and mutation analyses and succeeded in identifying the residues of Rab35 and centaurin-β2 that are crucial for formation of a Rab35·centaurin-β2 complex. We found that two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are responsible for binding centaurin-β2 and that the same residues are dispensable for Rab35 recognition by other Rab35-binding proteins. We also determined the minimal Rab35-binding site of centaurin-β2 and identified two asparagine residues (Asn-610 and Asn-691) in the Rab35-binding site as key residues for its specific Rab35 recognition. We further showed by knockdown-rescue approaches that neither a centaurin-β2 binding-deficient Rab35(T76S/T81A) mutant nor a Rab35 binding-deficient centaurin-β2(N610A/N691A) mutant supported neurite outgrowth of PC12 cells, thereby demonstrating the functional significance of the Rab35/centaurin-β2 interaction during neurite outgrowth of PC12 cells.
authors.affiliation: From the Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
authors.forename: Kan
authors.initials: K
authors.lastname: Etoh
authors.affiliation: From the Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan nori@m.tohoku.ac.jp.
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Fukuda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016501:Neurites; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: C575760:ACAP2 protein, rat; D017931:DNA Primers; D020690:GTPase-Activating Proteins; C575759:Rab35 protein, rat; D020691:rab GTP-Binding Proteins
keywords: Ankyrin; Membrane Trafficking; Neurite Outgrowth; Rab; Site-directed Mutagenesis; Small GTPase