van der Spek, SJF; Koopmans, F; Paliukhovich, I; Ramsden, SL; Harvey, K; Harvey, RJ; Smit, AB; van der Spek, SJF; Koopmans, F; Paliukhovich, I; Ramsden, SL; Harvey, K; Harvey, RJ; Smit, AB; Li, KW; - view fewer (2020) Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry. Proteomics 10.1002/pmic.201900403. (In press).

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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, we provide 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. When the affinity‐isolated GlyR complexes were fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β‐GPHN appeared as the most abundant complex with a molecular weight of approximately 1 MDa, and GlyR α1β‐GPHN‐IQSEC3 as a minor protein complex of approximately 1.2 MDa. A third GlyR α1β‐GPHN‐IQSEC2 complex existed at the lowest amount with a mass similar to the IQSEC3‐containing complex. Using yeast two‐hybrid we demonstrate that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N‐terminal of the IQSEC3 IQ‐like domain. Our 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.

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