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

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

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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.

Type: Article
Title: Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry.
Location: Germany
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/pmic.201900403
Publisher version: https://doi.org/10.1002/pmic.201900403
Language: English
Additional information: © 2020 The Authors. Proteomics published by Wiley‐VCH Verlag GmbH & Co. KGaA This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Keywords: IQSEC3, blue native gel electrophoresis, glycine receptor, protein complex, proteomics
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > Pharmacology
URI: https://discovery.ucl.ac.uk/id/eprint/10090506
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