Splice-specific Glycine Receptor Binding, Folding, and Phosphorylation of the Scaffolding Protein Gephyrin
| Autor: | Jens Herweg, Guenter Schwarz |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Scaffold protein
Protein Folding Glycine Receptor Binding Plasma protein binding Spodoptera Biochemistry Receptors Glycine Protein structure Neurobiology Receptors GABA Animals Phosphorylation Protein Structure Quaternary Molecular Biology Glycine receptor Cells Cultured Gephyrin biology Membrane Proteins Neural Inhibition Cell Biology Protein Structure Tertiary Rats Alternative Splicing Synapses Biophysics biology.protein Protein folding Carrier Proteins Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 287:12645-12656 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m112.341826 |
| Popis: | The multimeric scaffolding protein gephyrin forms post-synaptic clusters at inhibitory sites, thereby anchoring inhibitory glycine (GlyR) and subsets of γ-aminobutyric acid type A (GABAA) receptors. Gephyrin is composed of three domains, the conserved N-terminal G- and C-terminal E-domain, connected by the central (C-) domain. In this study we investigated the oligomerization, folding and stability, GlyR β-loop binding, and phosphorylation of three gephyrin splice variants (Geph, Geph-C3, Geph-C4) after expression and purification from insect cells (Sf9). In contrast to Escherichia coli-derived trimeric gephyrin, we found that Sf9 gephyrins form hexamers as basic oligomeric form. In the case of Geph and Geph-C4, also high-oligomeric forms (∼900 kDa) were isolated. Partial proteolysis revealed a compact folding of the Gephyrin G and C domain in one complex, whereas a much lower stability for the E domain was found. After GlyR β-loop binding, the stability of the E domain increased in Geph and Geph-C4 significantly. In contrast, the E domain in Geph-C3 is less stable and binds the GlyR β-loop with one order of magnitude lower affinity. Finally, we identified 18 novel phosphorylation sites in gephyrin, of which all except one are located within the C domain. We propose two models for the domain arrangement in hexameric gephyrin based on the oligomerization of either the E or C domains, with the latter being crucial for the regulation of gephyrin clustering. |
| Databáze: | OpenAIRE |
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