AbstractThe brainspecific GDP/GTP exchange factor collybistin interacts with the receptoranchoring protein gephyrin and activates the Rholike GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with heterooligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pulldown assays and in the yeast twohybrid system. Sitedirected mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyRβsubunit binding motif. Our data are consistent with collybistingephyrin interactions occuring during inhibitory postsynaptic membrane formation.