Synaptic adhesion molecules regulate multiple steps of synapse formation and maturation. The great diversity of neuronal synapses predicts the presence of a large number of adhesion molecules that control synapse formation through trans-synaptic and heterophilic adhesion. We identified a previously unknown trans-synaptic interaction between netrin-G ligand-3 (NGL-3), a postsynaptic density (PSD) 95-interacting postsynaptic adhesion molecule, and leukocyte common antigen-related (LAR), a receptor protein tyrosine phosphatase. NGL-3 and LAR expressed in heterologous cells induced pre- and postsynaptic differentiation in contacting axons and dendrites of cocultured rat hippocampal neurons, respectively. Neuronal overexpression of NGL-3 increased presynaptic contacts on dendrites of transfected neurons. Direct aggregation of NGL-3 on dendrites induced coclustering of excitatory postsynaptic proteins. Knockdown of NGL-3 reduced the number and function of excitatory synapses. Competitive inhibition by soluble LAR reduced NGL-3-induced presynaptic differentiation. These results suggest that the trans-synaptic adhesion between NGL-3 and LAR regulates excitatory synapse formation in a bidirectional manner.

Liprin-␣/SYD-2 is a family of multidomain proteins with four known isoforms. One of the reported functions of liprin-␣ is to regulate the development of presynaptic active zones, but the underlying mechanism is poorly understood. Here we report that liprin-␣ directly interacts with the ERC (ELKS-Rab6-interacting protein-CAST) family of proteins, members of which are known to bind RIMs, the active zone proteins that regulate neurotransmitter release. In vitro results indicate that ERC2/CAST, an active zone-specific isoform, interacts with all of the known isoforms of liprin-␣ and that liprin-␣1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-␣1 in cultured neurons and forms a complex with liprin-␣1 in brain. Liprin-␣1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-␣1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-␣-binding region, is sufficient for the synaptic localization of liprin-␣1 while the second half is not. These results suggest that the interaction between ERC2 and liprin-␣ may be involved in the presynaptic localization of liprin-␣ and the molecular organization of presynaptic active zones.

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and ␤PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of ␤PIX. Shank colocalizes with ␤PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with ␤PIX and ␤PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of ␤PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits ␤PIX and PAK to spines for the regulation of postsynaptic structure.Dendritic spines are actin-rich morphological specializations in neurons that mediate most excitatory synaptic transmission (1-3). The postsynaptic density (PSD) 1 is a microscopic structure within dendritic spines that is associated with the postsynaptic membrane and contains a variety of scaffolding and signaling proteins (4, 5).The Shank/ProSAP/SSTRIP family of multidomain proteins (Shank1, Shank2, and Shank3) plays important roles in organizing the PSD (6, 7). Shank is a relatively large protein (ϳ200 kDa) and contains various protein interaction domains including, from the N terminus, ankyrin repeats, an SH3 domain, a PDZ domain, a long (Ͼ1000 aa residues) proline-rich region and a SAM domain. The ankyrin repeats interact with ␣-fodrin, an actin-regulating protein, and Sharpin, a protein implicated in Shank multimerization (8, 9). The Shank PDZ domain interacts with the GKAP/SAPAP family of synaptic scaffold proteins and various membrane proteins including the calciumindependent receptor for latrotoxin, somatostatin receptors, and metabotropic glutamate receptors (10 -16). The long proline-rich region of Shank associates with IRSp53 (an insulin receptor tyrosine kinase substrate protein), Homer (an immediate early gene product that binds the group I metabotropic receptors and inositol 1,4,5-trisphosphate receptors), dynamin (a GTPase that regulates endocytosis), and cortactin (a regulator of the cortical actin cytoskeleton) (16 -20). The C-terminal SAM domain mediates multimerization of Shank proteins (10). There are several splice variants of Shank with alternative translational start and stop codons, suggesting that the Shank protein interactions are regulated by alternative splicing (11,12,21,22).Functionally, Shank is involved in the morphogenesis of dendritic spines (3, 23). Overexpression of Shank proteins promotes the maturation of spines in cultured neurons (24). The enhanced spine maturation by Shank requires the interaction of Shank with Homer, a protein that binds to metabotropic glutamate receptors and inositol 1,4,5-trisphosphate receptors (16). In addition, expression of ...

The cytoskeletal matrix assembled at active zones (CAZ) is implicated in defining neurotransmitter release sites. However, little is known about the molecular mechanisms by which the CAZ is organized. Here we report a novel interaction between Piccolo, a core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for ADP-ribosylation factor small GTPases. A small region (ϳ150 amino acid residues) in Piccolo, which is not conserved in the closely related CAZ protein Bassoon, mediates a direct interaction with the Spa2 homology domain (SHD) domain of GIT1. Piccolo and GIT1 colocalize at synaptic sites in cultured neurons. In brain, Piccolo forms a complex with GIT1 and various GIT-associated proteins, including ␤PIX, focal adhesion kinase, liprin-␣, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, ␤PIX, and focal adhesion kinase, suggesting that these proteins bind to the SHD by different mechanisms. Intriguingly, GIT proteins form homo-and heteromultimers through their C-terminal G-protein-coupled receptor kinase-binding domain in a tail-to-tail fashion. This multimerization enables GIT1 to simultaneously interact with multiple SHDbinding proteins including Piccolo and ␤PIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ.

Stargazin is a transmembrane protein that interacts with AMPARs 1 and regulates their synaptic targeting (1, 2). The stargazer, a spontaneous mutant mouse (3) with defects in the stargazin gene (Cacng2) (4), displays an absence seizure (also known as petit-mal or spike-wave) and, as the name implies, a head-tossing movement, probably because of a defect in the vestibular system (3). In addition, stargazer mice develop an ataxic gait (3) and severe impairment in classical eye-blink conditioning (5), probably because of a cerebellar malfunction. Both mRNA and protein levels of brain-derived neurotrophic factor are selectively reduced in cerebellar granule cells of stargazer mice (5, 6). Stargazin, a protein with a calculated molecular mass of 36 kDa, contains four putative transmembrane domains and a cytosolic C terminus, and its primary structure is closely related to that of the ␥ subunits of voltagegated calcium channels (7-10). Stargazin (or ␥-2) associates with neuronal calcium channel subunits in vivo (11) and inhibits calcium channel activity by increasing steady-state inactivation (4,7,11,12).The functional association between stargazin and AMPAR was initially ascertained by the observation that postsynaptic AMPAR currents are selectively impaired in cerebellar granule cells of stargazer mice (13). A subsequent study revealed that stargazin mediates synaptic targeting of AMPARs by two distinct mechanisms (14). Stargazin initially interacts with AMPARs and assists their translocation to the extrasynaptic surface membrane. Next, the AMPAR-stargazin complex is targeted to synaptic sites by binding to PSD-95 and related PDZ proteins. In support of this hypothesis, a stargazin mutant lacking the last four residues (stargazin⌬C) rescues extrasynaptic but not synaptic AMPAR currents in cerebellar granule cells of stargazer mice (14). However, little is known about whether the stargazin-mediated synaptic targeting of AMPARs is regulated and, if so, what these regulatory mechanisms involve.The C terminus of stargazin contains the end sequence RRT-TPV, which belongs to the class I PDZ-binding motif, (S/T)XV (S/T, Ser or Thr; X, any aa residue; V, hydrophobic residue) (15-17). Interestingly, the RRTT sequence of the C terminus additionally corresponds to the consensus sequence for phosphorylation by PKA, (R/K)(R/X)X(S/T), suggesting that Thr at the Ϫ2 position (RRTTPV, designated T321) is phosphorylated by PKA. The crystal structure of the PDZ3 domain of PSD-95 (class I) complexed with the C terminus of CRIPT, a PSD-95-binding protein that ends with the QTSV sequence (18), reveals that the Thr residue at the Ϫ2 position interacts with His-372 of PDZ3 (15). Specifically, the hydroxyl oxygen of the Thr forms a hydrogen bond with the N-3 nitrogen of His-372. Therefore, phosphorylation of T321 at the stargazin C terminus may weaken the interaction between stargazin and the PDZ domains of PSD-95. Consistently, earlier data demonstrate that phosphorylation of the Ser residue at the Ϫ2 position of Kir2.3 (an inward rectifie...

Mutation in KIF1B, a kinesin superfamily motor protein, causes a peripheral neuropathy known as Charcot-Marie-Tooth disease type 2A (CMT2A). Little is known, however, about how a defective KIF1B gene leads to CMT2A. Here we report that KIF1Balpha, one of the two splice variants of KIF1B, directly interacts through its C-terminal postsynaptic density-95 (PSD-95)/discs large/zona occludens (PDZ) domain-binding motif with PDZ proteins including PSD-95/synapse-associated protein-90 (SAP90), SAP97, and synaptic scaffolding molecule (S-SCAM)-90 (SAP90). KIF1Balpha selectively interacts with PSD-95, SAP97, and S-SCAM in yeast two-hybrid, pull-down, and in vivo coimmunoprecipitation experiments. KIF1Balpha, SAP97, and S-SCAM are widely distributed to both dendrites and axons of cultured neurons and are enriched in the small membrane fraction of the brain. In the flotation assay, KIF1Balpha cofractionates and coimmunoprecipitates with PSD-95, SAP97, and S-SCAM. These results suggest that the PSD-95 family proteins and S-SCAM have a novel function as KIF1Balpha receptors, linking KIF1Balpha to its specific cargos, and are involved in peripheral neuropathies.

Motor proteins not actively involved in transporting cargoes should remain inactive at sites of cargo loading to save energy and remain available for loading. KIF1A/ Unc104 is a monomeric kinesin known to dimerize into a processive motor at high protein concentrations. However, the molecular mechanisms underlying monomer stabilization and monomer-to-dimer transition are not well understood. Here, we report an intramolecular interaction in KIF1A between the forkhead-associated (FHA) domain and a coiled-coil domain (CC2) immediately following the FHA domain. Disrupting this interaction by point mutations in the FHA or CC2 domains leads to a dramatic accumulation of KIF1A in the periphery of living cultured neurons and an enhancement of the microtubule (MT) binding and self-multimerization of KIF1A. In addition, point mutations causing rigidity in the predicted flexible hinge disrupt the intramolecular FHA-CC2 interaction and increase MT binding and peripheral accumulation of KIF1A. These results suggest that the intramolecular FHA-CC2 interaction negatively regulates KIF1A activity by inhibiting MT binding and dimerization of KIF1A, and point to a novel role of the FHA domain in the regulation of kinesin motors.

KIF1A is a kinesin motor known to transport synaptic vesicle precursors in neuronal axons, but little is known about whether KIF1A mediates fast and processive axonal transport in vivo. By monitoring movements of EGFP-labeled KIF1A in living cultured hippocampal neurons, we determined the characteristics of KIF1A movements. KIF1A particles moved anterogradely along the neurites with an average velocity of 1.0 m/s. The movements of KIF1A were highly processive, with an average duration of persistent anterograde movement of 11 s. Some KIF1A particles (17%) exhibited retrograde movements of 0.72 m/s, although overall particle movement was in the anterograde direction. The anterograde movement of KIF1A, however, did not lead to a detectable accumulation of KIF1A in the periphery of neurons, suggesting that there are mechanisms inhibiting the peripheral accumulation of KIF1A. These results suggest that KIF1A mediates neuronal transport at a high velocity and processivity in vivo.