title: Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin.
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(A)R) subtypes. We report a direct interaction between the GABA(A)R 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(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R 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(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R 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(A)R 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 GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.
authors: Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University Vienna, Spitalgasse 4, 1090 Vienna, Austria.
authors: Verena
authors: V
authors: Tretter
authors: Bernd
authors: B
authors: Kerschner
authors: Ivan
authors: I
authors: Milenkovic
authors: Sarah L
authors: SL
authors: Ramsden
authors: Joachim
authors: J
authors: Ramerstorfer
authors: Leila
authors: L
authors: Saiepour
authors: Hans-Michael
authors: HM
authors: Maric
authors: Stephen J
authors: SJ
authors: Moss
authors: Hermann
authors: H
authors: Schindelin
authors: Robert J
authors: RJ
authors: Harvey
authors: Werner
authors: W
authors: Sieghart
authors: Kirsten
authors: K
authors: Harvey
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D059330:GABAergic Neurons / Q000166:cytology / Q000378:metabolism*; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D013569:Synapses / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D011963:Receptors, GABA-A; C073441:gephyrin