Splice-specific glycine receptor binding, folding, and phosphorylation of the scaffolding protein gephyrin.

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.