The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking

J Neurosci. 2011 Sep 28;31(39):13758-70. doi: 10.1523/JNEUROSCI.2649-11.2011.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / enzymology*
  • Cell Membrane / metabolism
  • Dopamine Plasma Membrane Transport Proteins / metabolism*
  • Glycoproteins / metabolism*
  • HEK293 Cells
  • Humans
  • Membrane Microdomains / enzymology*
  • Membrane Microdomains / metabolism
  • Mitochondrial Proteins / metabolism
  • Monomeric GTP-Binding Proteins / metabolism
  • Nerve Tissue Proteins / metabolism*
  • PC12 Cells
  • Protein Binding / physiology
  • Protein Kinase C / physiology*
  • Protein Transport / physiology
  • Rats
  • ras Proteins / metabolism

Substances

  • Dopamine Plasma Membrane Transport Proteins
  • Glycoproteins
  • Mitochondrial Proteins
  • Nerve Tissue Proteins
  • Rit2 protein, mouse
  • Protein Kinase C
  • RAB40AL protein, human
  • Monomeric GTP-Binding Proteins
  • RiT2 protein, human
  • ras Proteins