Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry

Sophie J.F. van der Spek, Frank Koopmans, Iryna Paliukhovich, Sarah L. Ramsden, Kirsten Harvey, Robert J. Harvey, August B. Smit, Ka Wan Li*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity-isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β-GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1β-GPHN-IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1β-GPHN-IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two-hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.

Original languageEnglish
Article number1900403
Pages (from-to)1-9
Number of pages9
JournalProteomics
Volume20
Issue number3-4
Early online date27 Jan 2020
DOIs
Publication statusPublished - 19 Feb 2020

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Keywords

  • blue native gel electrophoresis
  • glycine receptors
  • IQ motif and Sec7 domain 3
  • protein complexes
  • proteomics

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