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

Sophie J F Van Der Spek,Robert J Harvey,Frank Koopmans,Kirsten Harvey,Sarah L Ramsden,Iryna Paliukhovich,Ka Wan Li,August B Smit

doi:10.1002/pmic.201900403

Sophie J F Van Der Spek, Robert J Harvey + Show 6 more

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https://doi.org/10.1002/pmic.201900403

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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, 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.

Highlights

The glycine receptor (GlyR) is an anion-selective ligand-gated ion channel expressed mainly in the brain stem and spinal cord
In this study we examined the interactome of the major adult GlyR (α1β) subtype by immunoprecipitating the GlyR subunits GlyR α1, β and GPHN and identifying interacting proteins by mass spectrometry
For this we used a commercially available GlyRα1 antibody obtained from Genscript and three custom-made antibodies raised against GlyRα1, GlyRβ and GPHN, which recognised the bait proteins (Supplementary Fig. 1A, see Supplementary Materials and Methods)

Summary

Introduction

The glycine receptor (GlyR) is an anion-selective ligand-gated ion channel expressed mainly in the brain stem and spinal cord. The glycinergic synapse has a relatively simple postsynaptic density at the ultra-structural level [3]. It consists of multiple GlyRs, each composed of a pentamer of α1 and β subunits clustered by the synaptic scaffolding protein gephyrin (GPHN), and is thought to have limited ability for plastic change [3]. Recent interaction proteomics studies on ligand-gated ion channels and membrane receptors, such as GABAA and glutamate receptors, have revealed the presence of many protein interactors [4,5,6,7,8]. The relatively simple composition known of the GlyR complex (GlyR α1β-GPHN) may in part be explained by the low sensitive biochemical methods used in the previous studies, leaving additional interactors undetected.

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