Dataset Information

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.

SUBMITTER: Herweg J

PROVIDER: S-EPMC3339950 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Glycine and ?-aminobutyric acid (GABA) are the major determinants of inhibition in the central nervous system (CNS). These neurotransmitters target glycine and GABAA receptors, respectively, which both belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGICs). Interactions of the neurotransmitters with the cognate receptors result in receptor opening and a subsequent influx of chloride ions, which, in turn, leads to hyperpolarization of the membrane potential, thus counteracting excitatory stimuli. The majority of glycine receptors and a significant fraction of GABAA receptors (GABAARs) are recruited and anchored to the post-synaptic membrane by the central scaffolding protein gephyrin. This ?93 kDa moonlighting protein is structurally organized into an N-terminal G-domain (GephG) connected to a C-terminal E-domain (GephE) via a long unstructured linker. Both inhibitory neurotransmitter receptors interact via a short peptide motif located in the large cytoplasmic loop located in between transmembrane helices 3 and 4 (TM3-TM4) of the receptors with a universal receptor-binding epitope residing in GephE. Gephyrin engages in nearly identical interactions with the receptors at the N-terminal end of the peptide motif, and receptor-specific interaction toward the C-terminal region of the peptide. In addition to its receptor-anchoring function, gephyrin also interacts with a rather large collection of macromolecules including different cytoskeletal elements, thus acting as central scaffold at inhibitory post-synaptic specializations. Dysfunctions in receptor-mediated or gephyrin-mediated neurotransmission have been identified in various severe neurodevelopmental disorders. Although biochemical, cellular and electrophysiological studies have helped to understand the physiological and pharmacological roles of the receptors, recent high resolution structures of the receptors have strengthened our understanding of the receptors and their gating mechanisms. Besides that, multiple crystal structures of GephE in complex with receptor-derived peptides have shed light into receptor clustering by gephyrin at inhibitory post-synapses. This review will highlight recent biochemical and structural insights into gephyrin and the GlyRs as well as GABAA receptors, which provide a deeper understanding of the molecular machinery mediating inhibitory neurotransmission.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets