Journal article
Molecular basis of the γ-aminobutyric acid a receptor α3 subunit interaction with the clustering protein gephyrin
Journal of Biological Chemistry, Vol.286(43), pp.37702-37711
2011
Abstract
The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA A receptor (GABA AR) subtypes. We report a direct interaction between the GABA AR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA AR α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA AR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA ARs in recombinant systems. Interestingly, motifs responsible for interactions with GABA ARα2, GABA ARα3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA AR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA AR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μM and 0.2 μM, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Details
- Title
- Molecular basis of the γ-aminobutyric acid a receptor α3 subunit interaction with the clustering protein gephyrin
- Authors
- V Tretter (Author) - Medical University Vienna, AustriaB Kerschner (Author) - Medical University Vienna, AustriaI Milenkovic (Author) - Medical University Vienna, AustriaS L Ramsden (Author) - University College London, United KingdomJ Ramerstorfer (Author) - Medical University Vienna, AustriaL Saiepour (Author) - University College London, United KingdomH M Maric (Author) - University of Würzburg, GermanyStephen J Moss (Author) - Tufts University School of Medicine, United StatesH Schindelin (Author) - University of Würzburg, GermanyRobert J Harvey (Author) - University College London, United KingdomW Sieghart (Author) - Medical University Vienna, AustriaK Harvey (Author) - University College London, United Kingdom
- Publication details
- Journal of Biological Chemistry, Vol.286(43), pp.37702-37711
- Publisher
- American Society for Biochemistry and Molecular Biology, Inc.
- Date published
- 2011
- DOI
- 10.1074/jbc.M111.291336
- ISSN
- 0021-9258
- Copyright note
- Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc. The published version is reproduced here in accordance with the publisher's copyright policy
- Organisation Unit
- School of Health; University of the Sunshine Coast, Queensland; School of Health and Sport Sciences - Legacy; Centre for Bioinnovation; School of Health and Behavioural Sciences - Legacy
- Language
- English
- Record Identifier
- 99450426802621
- Output Type
- Journal article
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