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.
Highlights
Gephyrin is a postsynaptic scaffolding protein at inhibitory synapses and undergoes alternative splicing
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
In contrast to the well known gephyrin blobs seen in HEK293 cells [26, 29] or COS7 [33], expression of all three gephyrin variants in Sf9 cells showed diffuse staining of gephyrin in the cytosol with clear enrichment in the vicinity of the plasma membrane for gephyrin P1 (Geph) (Fig. 1A) and gephyrin C4 (Geph-C4) (Fig. 1B), whereas gephyrin C3 (Geph-C3) (Fig. 1C) was more diffuse in the cytosol
Summary
Gephyrin is a postsynaptic scaffolding protein at inhibitory synapses and undergoes alternative splicing. Splice-specific folding and stability, glycine receptor -loop binding, and phosphorylation of gephyrin were found. In this study we investigated the oligomerization, folding and stability, GlyR -loop binding, and phosphorylation of three gephyrin splice variants (Geph, Geph-C3, Geph-C4) after expression and purification from insect cells (Sf9). Crystal structures of the conserved G and E domains were determined and show tightly folded trimers [14, 15] and dimers [16], respectively This observed oligomerization behavior suggested a hexagonal lattice of gephyrin underneath the post-synaptic membrane [17]. We identified a serine residue within the binding motif of GlyR -loop that upon phosphorylation weakens gephyrin binding and thereby controls receptor scaffold interaction [21].
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