Gephyrin-mediated γ-Aminobutyric Acid Type A and Glycine Receptor Clustering Relies on a Common Binding Site

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 beta subunit (GlyR beta) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the alpha 1, alpha 2, and alpha 3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these alpha-subunits and compare them to the crystal structure of the gephyrin-GlyR beta complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR beta, gephyrin only loosely interacts with GABAAR alpha 2, whereas it has an intermediate affinity for the GABAAR alpha 1 and alpha 3 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 beta also abolished the GABA(A)R alpha 1 and alpha 3 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 alpha 2 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.