A combined transgenic proteomic analysis and regulated trafficking of neuroligin-2

J Biol Chem. 2014 Oct 17;289(42):29350-64. doi: 10.1074/jbc.M114.549279. Epub 2014 Sep 4.

Abstract

Synapses, the basic units of communication in the brain, require complex molecular machinery for neurotransmitter release and reception. Whereas numerous components of excitatory postsynaptic sites have been identified, relatively few proteins are known that function at inhibitory postsynaptic sites. One such component is neuroligin-2 (NL2), an inhibitory synapse-specific cell surface protein that functions in cell adhesion and synaptic organization via binding to neurexins. In this study, we used a transgenic tandem affinity purification and mass spectrometry strategy to isolate and characterize NL2-associated complexes. Complexes purified from brains of transgenic His6-FLAG-YFP-NL2 mice showed enrichment in the Gene Ontology terms cell-cell signaling and synaptic transmission relative to complexes purified from wild type mice as a negative control. In addition to expected components including GABA receptor subunits and gephyrin, several novel proteins were isolated in association with NL2. Based on the presence of multiple components involved in trafficking and endocytosis, we showed that NL2 undergoes dynamin-dependent endocytosis in response to soluble ligand and colocalizes with VPS35 retromer in endosomes. Inhibitory synapses in brain also present a particular challenge for imaging. Whereas excitatory synapses on spines can be imaged with a fluorescent cell fill, inhibitory synapses require a molecular tag. We find the His6-FLAG-YFP-NL2 to be a suitable tag, with the unamplified YFP signal localizing appropriately to inhibitory synapses in multiple brain regions including cortex, hippocampus, thalamus, and basal ganglia. Altogether, we characterize NL2-associated complexes, demonstrate regulated trafficking of NL2, and provide tools for further proteomic and imaging studies of inhibitory synapses.

Keywords: Adhesion; Dynamin; Endocytosis; GABAergic; Neurexin; Neuron; Retromer; Synapse; Trafficking; Transgenic.

Publication types

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

MeSH terms

  • Animals
  • Brain / metabolism
  • COS Cells
  • Cell Adhesion Molecules, Neuronal / metabolism*
  • Chlorocebus aethiops
  • Endocytosis
  • Hippocampus / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Nerve Tissue Proteins / metabolism*
  • Neural Inhibition / physiology
  • Neurons / metabolism
  • Protein Transport / genetics
  • Proteome
  • Proteomics / methods*
  • Synapses / metabolism*
  • Synaptic Transmission / physiology
  • Transgenes

Substances

  • Cell Adhesion Molecules, Neuronal
  • Nerve Tissue Proteins
  • Proteome
  • neuroligin 2