title: Splice-specific glycine receptor binding, folding, and phosphorylation of the scaffolding protein gephyrin.
abstract: 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.
authors.affiliation: Institute of Biochemistry, Department of Chemistry and Center for Molecular Medicine, University of Cologne, 50674 Cologne, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Herweg
authors.forename: Guenter
authors.initials: G
authors.lastname: Schwarz
mesh_terms: D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009433:Neural Inhibition / Q000502:physiology*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D018079:Receptors, GABA / Q000378:metabolism; D018009:Receptors, Glycine / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism; D013569:Synapses / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D018079:Receptors, GABA; D018009:Receptors, Glycine; C073441:gephyrin