title: The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking.
abstract: Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.
authors.affiliation: Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, Interdisciplinary Graduate Program, Graduate School of Biomedical Sciences, Graduate Program in Neuroscience, and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
authors.forename: Deanna M
authors.initials: DM
authors.lastname: Navaroli
authors.forename: Zachary H
authors.initials: ZH
authors.lastname: Stevens
authors.forename: Zeljko
authors.initials: Z
authors.lastname: Uzelac
authors.forename: Luke
authors.initials: L
authors.lastname: Gabriel
authors.forename: Michael J
authors.initials: MJ
authors.lastname: King
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Lifshitz
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Haley E
authors.initials: HE
authors.lastname: Melikian
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000201:enzymology* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D018631:ras Proteins / Q000378:metabolism
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D006023:Glycoproteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins; C089283:Rit2 protein, mouse; D011493:Protein Kinase C; C466300:RAB40AL protein, human; D020559:Monomeric GTP-Binding Proteins; C583559:RiT2 protein, human; D018631:ras Proteins