Gephyrin and collybistin are key components of GABA A receptor (GABA A R) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA A R subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA A R ␣2 and ␣3 subunit intracellular M3-M4 domain (but not ␣1, ␣4, ␣5, ␣6, ␤1-3, or ␥1-3) with gephyrin. Curiously, GABA A R ␣2, but not ␣3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA A R ␣2 also overlap at the start of the gephyrin E domain. This suggests that although GABA A R ␣3 interacts with gephyrin, GABA A R ␣2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA A R ␣2 and collybistin or GABA A R ␣2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA A R ␣2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA A R ␣2 is capable of "activating " collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA A R ␣2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA A R and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with ␣2 subunit containing GABA A Rs, but not GlyRs or other GABA A R subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA A Rs containing the ␣2 subunit.The clustering proteins gephyrin (1) and collybistin (2, 3) are thought to represent key players in the synaptic clustering of both glycine receptors (GlyRs) 4 and GABA A Rs. Studies using gephyrin knock-out mice or mRNA knockdown (4 -7) have shown a loss of postsynaptic clustering of GlyRs and GABA A Rs containing ␣2 and ␥2 subunits. However, certain GABA A R subtypes still appear to cluster in neurons lacking gephyrin (6 -9). Hence, although the majority of GlyRs are likely to be clustered by gephyrin (10), exactly which GABA A R subtypes are subject to gephyrin-dependent clustering remains unclear. What is certain is that the subcellular localization of gephyrin is dependent on certain GABA A R subtypes. For example, targeted deletion of the GABA A R ␣1, ␣3, and ␥2 subunit genes results in a loss of synaptic gephyrin and GABA A R clusters (11-15), with cytoplasmic gephyrin aggregates indicating disrupted synaptic targeting. Although the interaction of the GlyR ␤ subunit with the gephyrin E domain has been well characterized (3,16,17), the same cannot be said for GABA A Rs. Different mechanisms have been proposed to explain the complex interactions of GABA A Rs with gephyrin, including alternative splicing (18), intermediate acces...

Background: The molecular basis of GABA A receptor ␣3 subtype-specific synaptic localization is unknown. Results: GABA A R ␣3 interacts with the gephyrin E domain via defined intracellular motifs that partially overlap with glycine receptor binding determinants. Conclusion: GABA A R subtypes containing ␣3 are clustered at postsynaptic specializations via direct interactions with gephyrin. Significance: Distinct binding properties of GABA A R and GlyRs to gephyrin may govern mixed glycinergic/GABAergic transmission.