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.

FIGURE 1.

Subcellular localization of gephyrin splice variants in Sf9 cells. Sf9 cells were transfected with different gephyrin constructs and expressed for 48 h. Localization of Geph (A), Geph-C4 (B), and Geph-C3 (C) was visualized using mB311 antibodies (green) and laser scanning microscopy. D–F, nuclei were stained with Hoechst stain. Scale bars, 8 μm.

FIGURE 2.

Molecular mass determination of purified gephyrin oligomers. A–D, size exclusion chromatography of EcGeph-C4 from E. coli (A), Geph (B), Geph-C4 (C), and Geph-C3 (D) derived from Sf9 cells using a 20-ml Superose 6 column. Samples were derived from a first size exclusion run (supplemental Fig. S1, A–D) where hexameric (6xG, dashed line), high oligomeric (HOxG, bold line), and in the case of EcGeph-C4, trimeric (dotted line, A) fractions were taken. Molecular mass of gephyrin oligomers was determined by comparison to marker proteins of known size. Different oligomerization states are indicated by symbols: HOxG, high oligomer; 6xG, hexamer; 3xG, trimer. Purity of gephyrin oligomers was probed by SDS-PAGE (E). AU, absorbance units.

FIGURE 3.

Partial proteolysis of EcGeph-C4 and Geph-C4. E. coli-derived Geph-C4 (left) and Sf9 cell-derived Geph-C4 (right) were digested (6 μg each) with trypsin and separated by a 12% SDS-PAGE (A) together with equal amounts of untreated protein (−). Identification of gephyrin fragments was confirmed by Western blot using domain-specific antibodies detecting the G domain (B, puszta serum) and E-domain (C, m3B11).

FIGURE 4.

Thermal stability of gephyrin splice variants. Shown are DSC heating thermograms of gephyrin domain fragments (A–D) and gephyrin splice variants (E–H). In each experiment 20–50 μm protein was used. A–D, shown are purified gephyrin domains G (A), E (B), G-C (C), and E-C (D) expressed in E. coli. E–H, shown are EcGeph-C4 (E) and Sf9 cell-derived Geph (F), Geph-C4 (G), and Geph-C3 (H) in the absence (bold lines) and presence of 200–500 μm GlyR β-loop (+GlyRβ, dotted line). The molar heat capacity profiles were recorded from 20–90 ºC at a scan rate of 25 ºC/h. Quantitative values of the corresponding peak transition temperatures of gephyrin E domains (T_m1), gephyrin G domains (T_m2), and with additional GlyR β-loop (ΔT_m) are listed in Table 1.

FIGURE 5.

Binding affinity of gephyrin-glycine receptor β-loop interaction. The binding affinity of gephyrin splice variants with GlyR β-loop was measured by ITC. Binding isotherms of trimeric EcGeph-C4 (A, white square), hexaoligomeric and high oligomeric Geph (B, black and white circle), Geph-C4 (C, black and white triangle), and hexameric Geph-C3 (D, black square) titrated with the GlyR β-loop are shown. All experiments were carried out under the same conditions. For data analysis, the heat release of the first injection in each experiment was omitted. Measured binding enthalpies (supplemental Fig. S3) were plotted as a function of the molar ratio of GlyR β-loop to gephyrin. Determined binding parameters are summarized in Table 2.

FIGURE 6.

Phosphorylation analysis of Sf9 cell-expressed gephyrin. Peptide mass fingerprinting of Geph, Geph-C4 and Geph-C3 revealed several phosphorylation sites, all localized within the C-domain of gephyrin, except Thr-324. A, residues that were identified in one, two, or three splice variants are shaded in gray and printed in black, shaded in gray and printed in white, and shaded in black and printed in white, respectively. B–D, shown is a plot of the percent ratio of phosphorylated over non-phosphorylated peptides containing the indicated residues found in Geph (B), Geph-C4 (C), and Geph-C3 (D). The total number of peptides identified for the hexameric (6xG) and high oligomeric (HOxG) pool of gephyrin are plotted with solid and dashed lines, respectively.

FIGURE 7.

Models for the hexameric arrangement of gephyrin domains. G, C, and E domains are depicted in different shades. Shown are top and side views (90º rotated as indicated) of hexameric gephyrin either facing the E (A) or C domains (B) of each other. Note that based on our stability studies a interacting surface between G and C domain is depicted.