Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.

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IRSp53 is an essential intermediate between the activation of Rac and Cdc42GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and pairedpulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-D-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm.Rho GTPases such as Cdc42, Rac, and Rho control key events in neuronal cell biology, including the generation of neuronal polarity and morphology, establishment of dendritic spines, the generation of postsynaptic specializations and synaptic plasticity (1, 2). Specificity in these processes is thought to arise through control of different downstream targets which are recognized and activated by the active, GTP-bound forms of Rho family members. The insulin receptor substrate of 53 kDa (IRSp53) 3 is an essential mediator between activated Rac or Cdc42 and the formation of lamellipodia or filopodia, respectively. GTPase binding to IRSp53 enables interactions of its SH3 domain with downstream effectors WAVE2, Mena, Eps8, or N-WASP, all of which are known regulators of actin dynamics (3-6). In addition, the N-terminal IRSp53/missing in metastasis homology domain of IRSp53 assists in generating cellular protrusions by bundling actin filaments (5, 7, 8) and promoting membrane curvature (9, 10). Expression of IRSp53 is particularly high in the brain, and consequently IRSp53 contributes to the formation of dendritic spines in the cultured hippocampal neuron model (11).Via the SH3 domain and a C-terminal PDZ binding motif, IRSp53 also bridges postsynaptic shank and PSD-95 family members (11)(12)(13)(14). A significant enrichment in the postsynaptic density (PSD) of excitatory synapses suggests that Rac/Cdc42 signalin...

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/ IRSp53 pathway in postsynaptic sites is however, unclear. The generation of excitatory synapses in the central nervous system requires a complex assembly process in which elements of the postsynaptic receptor apparatus are assembled at postsynaptic sites on dendrites. In many cases, glutamatergic synapses are localized on the heads of dendritic spines (Harris and Kater 1994; see review by Hering and Sheng 2001). During maturation postsynaptic proteins accumulate at the synapse, as exemplified in several studies by the appearance of clusters of the postsynaptic marker PSD-95 (Friedman et al. 2000;Okabe et al. 2001). Via its PDZ domains, PSD-95 is one of the major anchoring proteins for postsynaptic transmitter receptors and ion channels (Kim et al. 1995;Kornau et al. 1995). Through an intricate network of protein interactions, a large protein complex of up to 100 proteins is assembled at the spine heads around the PSD-95/transmitter receptor complex (Husi et al. 2000;Walikonis et al. 2000;Li et al. 2004) which has been termed the postsynaptic density (PSD). The function of the PSD appears to be to physically link postsynaptic receptors to signalling molecules, and to provide stable attachment of the receptors to the actin-based cytoskeleton of the dendritic spine. Shank proteins (shank1-3, also known as SSTRIP, ProSAP, synamon or CortBP) constitute another group of postsynaptic scaffolding molecules which link transmitter receptors (Kreienkamp et al. 2000;Naisbitt et al. 1999;Yao et al. 1999;Zitzer et al. 1999) to actin binding proteins (Du et al. 1998;Boeckers et al. 2001;Okamoto et al. 2001). Overexpression of shank1 in neurones leads to enhanced maturation of dendritic spines . We and others have recently identified IRSp53 as an interaction partner for shank1 (Bockman et al. 2002;Soltau et al. 2002). A proline-rich region of shank1 binds to the SH3 domain of IRSp53 in a cdc42-regulated manner (Soltau et al. 2002). These data suggested that shank1 might be an effector molecule of cdc42 in an undefined regulatory pathway. Here, we show that IRSp53, via binding to a PDZ domain of the PSD-95 molecule, mediates the formation of a triple complex consisting of shank1 and PSD-95. Our data suggest that one result of cdc42/IRSp53 signalling is the regulated assembly of a macromolecular complex between shank and PSD-95 proteins.Received January 14, 2004; revised manuscript received March 19, 2004; accepted March 23, 2004. Address correspondence and reprint requests to Hans-Jürgen Kreienkamp, Institut für Zellbiochemie und klinische Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail: kreienkamp@uke.uni-hamburg.deAbbreviations used: IRSp53, insulin receptor su...