title: Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site.
abstract: Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.
authors: Rudolf Virchow Center for Experimental Biomedicine, Institute of Structural Biology, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors: Hans-Michael
authors: HM
authors: Maric
authors: Department of Neuroscience, Tufts University, Boston, Massachusetts 02111.
authors: Jayanta
authors: J
authors: Mukherjee
authors: Department of Biochemistry and Molecular Biology, Medical University Vienna/Center for Brain Research, Spitalgasse 4, 1090 Vienna, Austria.
authors: Verena
authors: V
authors: Tretter
authors: Department of Neuroscience, Tufts University, Boston, Massachusetts 02111.
authors: Stephen J
authors: SJ
authors: Moss
authors: Rudolf Virchow Center for Experimental Biomedicine, Institute of Structural Biology, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. Electronic address: hermann.schindelin@virchow.uni-wuerzburg.de.
authors: Hermann
authors: H
authors: Schindelin
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry; D016000:Cluster Analysis; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D018009:Receptors, Glycine / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism; D055366:Synaptic Potentials; D014443:Tyrosine / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D008024:Ligands; D008565:Membrane Proteins; D011963:Receptors, GABA-A; D018009:Receptors, Glycine; C073441:gephyrin; D014443:Tyrosine