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Gephyrin, the enigmatic organizer at GABAergic synapses.

Tretter V, Mukherjee J, Maric HM, Schindelin H, Sieghart W, Moss SJ - Front Cell Neurosci (2012)

Bottom Line: This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition.Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord.Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna Vienna, Austria.

ABSTRACT
GABA(A) receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABA(A) receptors have also been shown to directly interact with gephyrin and interaction sites have been identified and mapped within the intracellular loops of the GABA(A) receptor α1, α2, and α3 subunits. Gephyrin-binding to GABA(A) receptors seems to be at least one order of magnitude weaker than to glycine receptors (GlyRs) and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity.

No MeSH data available.


Related in: MedlinePlus

Structural determinants of gephyrin, interacting with GABAARs, GlyRs, and collybistin. (A) Crystal structure of the gephyrin E domain (GephE) complexed with GlyR β, modified for comparison with GABAAR α subunits and collybistin. The ribbon diagram of GephE is colored to highlight its subdomain architecture. The GephE-domain is complemented by a second monomer shown in grey, representing the dimer that usually forms in vitro. Identified binding sites for collybistin on gephyrin are shown in magenta and overlapping binding sites for GlyR β and GABAAR α subunits are shown in light blue. The GlyR β peptide as well as crucial interacting amino acids in GephE (D327, F330, P713) are shown in stick representation. (B) The major contributing residues to the interaction are conserved among GABAAR α3 and GlyR β and are marked in red. (C) Subdomain-structure of GephE and amino acid sequence of GephE harboring the main binding site to GABAAR α1, α2, and α3 subunits, GlyR β subunit and collybistin. Crucial residues for the interaction with receptors are highlighted in light blue (Asp327, Phe330), the overlapping collybistin binding site is shown in magenta. (D) The table compares homologous sequences crucial for gephyrin-binding in GlyR β and GABAAR α subunits from rat. Residues critically contributing to the interaction and conserved among all subtypes are shown in red. The dissociation constant KD indicates the binding strength as determined by isothermal titration calorimetry. (n.d. = not detectable).
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Figure 3: Structural determinants of gephyrin, interacting with GABAARs, GlyRs, and collybistin. (A) Crystal structure of the gephyrin E domain (GephE) complexed with GlyR β, modified for comparison with GABAAR α subunits and collybistin. The ribbon diagram of GephE is colored to highlight its subdomain architecture. The GephE-domain is complemented by a second monomer shown in grey, representing the dimer that usually forms in vitro. Identified binding sites for collybistin on gephyrin are shown in magenta and overlapping binding sites for GlyR β and GABAAR α subunits are shown in light blue. The GlyR β peptide as well as crucial interacting amino acids in GephE (D327, F330, P713) are shown in stick representation. (B) The major contributing residues to the interaction are conserved among GABAAR α3 and GlyR β and are marked in red. (C) Subdomain-structure of GephE and amino acid sequence of GephE harboring the main binding site to GABAAR α1, α2, and α3 subunits, GlyR β subunit and collybistin. Crucial residues for the interaction with receptors are highlighted in light blue (Asp327, Phe330), the overlapping collybistin binding site is shown in magenta. (D) The table compares homologous sequences crucial for gephyrin-binding in GlyR β and GABAAR α subunits from rat. Residues critically contributing to the interaction and conserved among all subtypes are shown in red. The dissociation constant KD indicates the binding strength as determined by isothermal titration calorimetry. (n.d. = not detectable).

Mentions: The binding site of the GlyR β subunit (GlyR β) on gephyrin was initially identified as a stretch of residues at the beginning of gephyrin's C-terminal E-domain (GephE, for domain structure see Figure 3C). In 2006, the GlyR β-loop was co-crystallized with the E-domain of gephyrin, showing that amino acids at the beginning and the end of the E-domain seem to be involved in GlyR β subunit binding (Schrader et al., 2004; Kim et al., 2006). Based on the structure of the GlyR β subunit-gephyrin complex and sequence similarities in the GABAAR α3 subunit we hypothesized, that binding of α3 to gephyrin very likely occurs in a similar three-dimensional binding pocket of the gephyrin E-domain. As a crystal structure is currently not available, we first roughly mapped the binding sites of GABAAR α2 and α3 subunits on gephyrin by using alanine mutagenesis in a yeast two-hybrid system (Saiepour et al., 2010; Tretter et al., 2011). The identified sequences for α2 (residues 325–343) and α3 (residues 325–334) overlap and include the critical aminoacids involved in GlyR β subunit binding (Asp327, Phe330) (Figure 3C). These results imply an evolutionarily conserved binding site for GABAAR α subunits and GlyR β subunit on gephyrin.


Gephyrin, the enigmatic organizer at GABAergic synapses.

Tretter V, Mukherjee J, Maric HM, Schindelin H, Sieghart W, Moss SJ - Front Cell Neurosci (2012)

Structural determinants of gephyrin, interacting with GABAARs, GlyRs, and collybistin. (A) Crystal structure of the gephyrin E domain (GephE) complexed with GlyR β, modified for comparison with GABAAR α subunits and collybistin. The ribbon diagram of GephE is colored to highlight its subdomain architecture. The GephE-domain is complemented by a second monomer shown in grey, representing the dimer that usually forms in vitro. Identified binding sites for collybistin on gephyrin are shown in magenta and overlapping binding sites for GlyR β and GABAAR α subunits are shown in light blue. The GlyR β peptide as well as crucial interacting amino acids in GephE (D327, F330, P713) are shown in stick representation. (B) The major contributing residues to the interaction are conserved among GABAAR α3 and GlyR β and are marked in red. (C) Subdomain-structure of GephE and amino acid sequence of GephE harboring the main binding site to GABAAR α1, α2, and α3 subunits, GlyR β subunit and collybistin. Crucial residues for the interaction with receptors are highlighted in light blue (Asp327, Phe330), the overlapping collybistin binding site is shown in magenta. (D) The table compares homologous sequences crucial for gephyrin-binding in GlyR β and GABAAR α subunits from rat. Residues critically contributing to the interaction and conserved among all subtypes are shown in red. The dissociation constant KD indicates the binding strength as determined by isothermal titration calorimetry. (n.d. = not detectable).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Structural determinants of gephyrin, interacting with GABAARs, GlyRs, and collybistin. (A) Crystal structure of the gephyrin E domain (GephE) complexed with GlyR β, modified for comparison with GABAAR α subunits and collybistin. The ribbon diagram of GephE is colored to highlight its subdomain architecture. The GephE-domain is complemented by a second monomer shown in grey, representing the dimer that usually forms in vitro. Identified binding sites for collybistin on gephyrin are shown in magenta and overlapping binding sites for GlyR β and GABAAR α subunits are shown in light blue. The GlyR β peptide as well as crucial interacting amino acids in GephE (D327, F330, P713) are shown in stick representation. (B) The major contributing residues to the interaction are conserved among GABAAR α3 and GlyR β and are marked in red. (C) Subdomain-structure of GephE and amino acid sequence of GephE harboring the main binding site to GABAAR α1, α2, and α3 subunits, GlyR β subunit and collybistin. Crucial residues for the interaction with receptors are highlighted in light blue (Asp327, Phe330), the overlapping collybistin binding site is shown in magenta. (D) The table compares homologous sequences crucial for gephyrin-binding in GlyR β and GABAAR α subunits from rat. Residues critically contributing to the interaction and conserved among all subtypes are shown in red. The dissociation constant KD indicates the binding strength as determined by isothermal titration calorimetry. (n.d. = not detectable).
Mentions: The binding site of the GlyR β subunit (GlyR β) on gephyrin was initially identified as a stretch of residues at the beginning of gephyrin's C-terminal E-domain (GephE, for domain structure see Figure 3C). In 2006, the GlyR β-loop was co-crystallized with the E-domain of gephyrin, showing that amino acids at the beginning and the end of the E-domain seem to be involved in GlyR β subunit binding (Schrader et al., 2004; Kim et al., 2006). Based on the structure of the GlyR β subunit-gephyrin complex and sequence similarities in the GABAAR α3 subunit we hypothesized, that binding of α3 to gephyrin very likely occurs in a similar three-dimensional binding pocket of the gephyrin E-domain. As a crystal structure is currently not available, we first roughly mapped the binding sites of GABAAR α2 and α3 subunits on gephyrin by using alanine mutagenesis in a yeast two-hybrid system (Saiepour et al., 2010; Tretter et al., 2011). The identified sequences for α2 (residues 325–343) and α3 (residues 325–334) overlap and include the critical aminoacids involved in GlyR β subunit binding (Asp327, Phe330) (Figure 3C). These results imply an evolutionarily conserved binding site for GABAAR α subunits and GlyR β subunit on gephyrin.

Bottom Line: This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition.Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord.Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna Vienna, Austria.

ABSTRACT
GABA(A) receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABA(A) receptors have also been shown to directly interact with gephyrin and interaction sites have been identified and mapped within the intracellular loops of the GABA(A) receptor α1, α2, and α3 subunits. Gephyrin-binding to GABA(A) receptors seems to be at least one order of magnitude weaker than to glycine receptors (GlyRs) and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity.

No MeSH data available.


Related in: MedlinePlus