Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission

In many brain regions, gephyrin and GABA_A receptor clustering at developing inhibitory synapses depends on the guanine nucleotide exchange factor collybistin (Cb). The vast majority of Cb splice variants contain an autoinhibitory src homology 3 domain, and several synaptic proteins are known to bind to this SH3 domain and to thereby activate gephyrin clustering. However, many functional GABAergic synapses form independently of the known Cb-activating proteins, indicating that additional Cb activators must exist. Here we show that the small Rho-like GTPase TC10 stimulates Cb-dependent gephyrin clustering by binding in its active, GTP-bound state to the pleckstrin homology domain of Cb. Overexpression of a constitutively active TC10 variant in neurons causes an increase in the density of synaptic gephyrin clusters and mean miniature inhibitory postsynaptic current amplitudes, whereas a dominant negative TC10 variant has opposite effects. The enhancement of Cb-induced gephyrin clustering by GTP-TC10 does not depend on the guanine nucleotide exchange activity of Cb but involves an interaction that resembles reported interactions of other small GTPases with their effectors. Our data indicate that GTP-TC10 activates the major src homology 3 domain-containing Cb variants by relieving autoinhibition and thus define an alternative GTPase-driven signaling pathway in the genesis of inhibitory synapses.Chemical synaptic transmission between neurons requires the tight packing of ionotropic neurotransmitter receptors in the postsynaptic plasma membrane. Core components of many inhibitory GABAergic postsynapses are the cell adhesion protein neuroligin 2 (NL2), the scaffolding protein gephyrin, the guanine nucleotide exchange factor (GEF) collybistin (Cb), and GABA_A receptors (GABA_ARs) (1, 2). The assembly of such GABAergic postsynapses is triggered by the interaction of NL2 with the src homology 3 (SH3) domain of Cb. This leads to the activation of Cb, which is otherwise autoinhibited by intramolecular interactions of its SH3 domain with the Dbl homology (DH) and pleckstrin homology (PH) domains, followed by membrane recruitment of Cb and synaptic accumulation of gephyrin and GABA_ARs (3). However, this NL2/Cb/gephyrin/GABA_AR interaction cascade cannot account for the formation of all GABAergic synapses, because in the hippocampus of NL2 KO mice gephyrin and GABA_AR clusters are lost only from perisomatic regions of CA1 pyramidal neurons (3). In contrast, deletion of Cb leads to a loss of gephyrin from both perisomatic and dendritic postsynapses (4). Thus, the formation of a substantial subset of GABAergic postsynapses must be regulated by Cb-interacting proteins other than NL2.Another class of Cb interaction partners with a potential role in gephyrin clustering are small Rho-like GTPases. They regulate many fundamental cellular processes, including actin cytoskeleton rearrangements (5), and the actin cytoskeleton plays an important role in the formation of inhibitory postsynapses, particularly at early stages of synapse formation (6, 7). The small GTPase Cdc42 is an established Cb substrate (8–10), and a recent analysis of 12 Rho-like GTPases identified Cdc42 as the only family member that can be activated in vitro by the human Cb ortholog hPem2 (11). However, Cdc42 expression is not required for gephyrin and GABA_AR clustering at postsynapses, indicating that Cb may regulate cytoskeleton remodeling by activating other Rho-like GTPases (10). The small GTPase most closely related to Cdc42 is TC10. Its sequence [67.4% amino acid identity (12)] and structure (13) are similar to those of Cdc42, it shares common cellular functions and effectors with Cdc42 (14), and profilin, an actin and gephyrin binding protein (15, 16), is an effector of TC10 (14). In contrast to Cdc42, which is ubiquitously expressed in the mammalian brain, the expression of TC10 is limited to specific areas, including the CA1 region of the hippocampus (17), where the most prominent reduction in gephyrin and GABA_AR clustering is observed in Cb KO mice (4). Here we provide evidence for an effector-type binding of GTP-TC10 to the PH domain of Cb that results in Cb activation, triggers synaptic gephyrin clustering, and enhances GABAergic neurotransmission.