Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry
| Autor: | August B. Smit, Kirsten Harvey, Frank Koopmans, Robert J. Harvey, Iryna Paliukhovich, Sarah L. Ramsden, Ka Wan Li, Sophie J. F. van der Spek |
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| Přispěvatelé: | Molecular and Cellular Neurobiology, Amsterdam Neuroscience - Cellular & Molecular Mechanisms, Center for Neurogenomics and Cognitive Research, AIMMS |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Scaffold protein
Electrophoresis Proteome Immunoprecipitation Inhibitory postsynaptic potential Proteomics Biochemistry 03 medical and health sciences Receptors Glycine proteomics Tandem Mass Spectrometry Animals Guanine Nucleotide Exchange Factors Humans Amino Acid Sequence Molecular Biology Glycine receptor 030304 developmental biology Neurons 0303 health sciences protein complexes Gephyrin biology Chemistry glycine receptors 030302 biochemistry & molecular biology Cell Membrane Membrane Proteins blue native gel electrophoresis IQ motif and Sec7 domain 3 Protein Subunits G-domain Synapses Biophysics biology.protein Electrophoresis Polyacrylamide Gel Function (biology) Protein Binding |
| Zdroj: | Proteomics, 20(3-4):1900403, 1-9. Wiley-VCH Verlag van der Spek, S J F, Koopmans, F, Paliukhovich, I, Ramsden, S L, Harvey, K, Harvey, R J, Smit, A B & Li, K W 2020, ' Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry ', Proteomics, vol. 20, no. 3-4, 1900403, pp. 1-9 . https://doi.org/10.1002/pmic.201900403 |
| ISSN: | 1615-9853 |
| DOI: | 10.1002/pmic.201900403 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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