Seho Kim

NGL family PSD-95–interacting adhesion molecules regulate excitatory synapse formation

Synaptic cell adhesion molecules (CAMs) regulate synapse formation through their trans-synaptic and heterophilic adhesion. Here we show that postsynaptic netrin-G ligand (NGL) CAMs associate with netrin-G CAMs in an isoform-specific manner and, through their cytosolic tail, with the abundant postsynaptic scaffold postsynaptic density–95 (PSD-95). Overexpression of NGL-2 in cultured rat neurons increased the number of PSD-95–positive dendritic protrusions. NGL-2 located on heterologous cells or beads induced functional presynaptic differentiation in contacting neurites. Direct aggregation of NGL-2 on the surface membrane of dendrites induced the clustering of excitatory postsynaptic proteins. Competitive inhibition by soluble NGL-2 reduced the number of excitatory synapses. NGL-2 knockdown reduced excitatory, but not inhibitory, synapse numbers and currents. These results suggest that NGL regulates the formation of excitatory synapses.

Trans-synaptic adhesion between NGL-3 and LAR regulates the formation of excitatory synapses.

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.

Regulation of Dendritic Spine Morphogenesis by Insulin Receptor Substrate 53, a Downstream Effector of Rac1 and Cdc42 Small GTPases

The small GTPases Rac1 and Cdc42 are key regulators of the morphogenesis of actin-rich dendritic spines in neurons. However, little is known about how activated Rac1/Cdc42 regulates dendritic spines. Insulin receptor substrate 53 (IRSp53), which is highly expressed in the postsynaptic density (PSD), is known to link activated Rac1/Cdc42 to downstream effectors for actin regulation in non-neural cells. Here, we report that IRSp53 interacts with two specific members of the PSD-95 family, PSD-95 and chapsyn-110/PSD-93, in brain. An IRSp53 mutant lacking the C-terminal PSD-95-binding motif shows significant loss of synaptic localization in cultured neurons. Overexpression of IRSp53 in cultured neurons increases the density of dendritic spines but does not affect their length or width. Conversely, short-interfering RNA-mediated knock-down of IRSp53 reduces the density, length, and width of spines. In addition, the density and size of spines are decreased by a dominant-negative IRSp53 with a point mutation in the Src homology 3 (SH3) domain and a dominant-negative proline-rich region of WAVE2 (Wiskott-Aldrich syndrome protein family Verprolin-homologous protein), a downstream effector of IRSp53 that binds to the SH3 domain of IRSp53. These results suggest that PSD-95 interaction is an important determinant of synaptic IRSp53 localization and that the SH3 domain of IRSp53 links activated Rac1/Cdc42 to downstream effectors for the regulation of spine morphogenesis.

SALM Synaptic Cell Adhesion-like Molecules Regulate the Differentiation of Excitatory Synapses

Synaptic cell adhesion molecules (CAMs) are known to play key roles in various aspects of synaptic structures and functions, including early differentiation, maintenance, and plasticity. We herein report the identification of a family of cell adhesion-like molecules termed SALM that interacts with the abundant postsynaptic density (PSD) protein PSD-95. SALM2, a SALM isoform, distributes to excitatory, but not inhibitory, synaptic sites. Overexpression of SALM2 increases the number of excitatory synapses and dendritic spines. Mislocalized expression of SALM2 disrupts excitatory synapses and dendritic spines. Bead-induced direct aggregation of SALM2 results in coclustering of PSD-95 and other postsynaptic proteins, including GKAP and AMPA receptors. Knockdown of SALM2 by RNA interference reduces the number of excitatory synapses and dendritic spines and the frequency, but not amplitude, of miniature excitatory postsynaptic currents. These results suggest that SALM2 is an important regulator of the differentiation of excitatory synapses.

An intramolecular interaction between the FHA domain and a coiled coil negatively regulates the kinesin motor KIF1A

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.

Human papillomavirus type 16 E6 protein interacts with cystic fibrosis transmembrane regulator-associated ligand and promotes E6-associated protein-mediated ubiquitination and proteasomal degradation

The PDZ proteins such as hDLG, hScrib and MAGIs function as the membrane-associated protein scaffolds and have been shown to interact with the high-risk human papillomavirus (HPV) E6s. In this report, we identify a Golgi-associated PDZ protein, cystic fibrosis transmembrane regulator-associated ligand (CAL) as a cellular target of HPV16 E6 by the proteomic approach. The carboxy-terminal PDZ-binding motif of HPV16 E6 specifically interacts with the PDZ domain of CAL, and the interaction enhances proteasome-mediated degradation of CAL. HPV16 E6 interacts with CAL more strongly and degrades it better than HPV18 E6 owing to the more compatible PDZ-binding motif. CAL is ubiquitinated by the E6/E6-associated protein (E6AP) complex or by E6AP alone, albeit less efficiently, which indicates that it could be a normal target of E6AP. Although it downregulates CAL at the transcript level, small interfering RNA-induced depletion of HPV16 E6 in Caski cells stabilizes CAL at the protein level, suggesting that HPV16 E6 mediates the proteasomal degradation of CAL in HPV-positive cervical cancer cells. HPV16 E6 may tightly regulate the vesicular trafficking processes by interacting with CAL, and such a modification can contribute to the development of cervical cancer.

Regulation of Dendritic Spines, Spatial Memory, and Embryonic Development by the TANC Family of PSD-95-Interacting Proteins

PSD-95 (postsynaptic density-95) is thought to play important roles in the regulation of dendritic spines and excitatory synapses, but the underlying mechanisms have not been fully elucidated. TANC1 is a PSD-95-interacting synaptic protein that contains multiple domains for protein-protein interactions but whose function is not well understood. In the present study, we provide evidence that TANC1 and its close relative TANC2 regulate dendritic spines and excitatory synapses. Overexpression of TANC1 and TANC2 in cultured neurons increases the density of dendritic spines and excitatory synapses in a manner that requires the PDZ (PSD-95/Dlg/ZO-1)-binding C termini of TANC proteins. TANC1-deficient mice exhibit reduced spine density in the CA3 region of the hippocampus, but not in the CA1 or dentate gyrus regions, and show impaired spatial memory. TANC2 deficiency, however, causes embryonic lethality. These results suggest that TANC1 is important for dendritic spine maintenance and spatial memory, and implicate TANC2 in embryonic development.

Regulation of neuritogenesis and synaptic transmission by msec7-1, a guanine nucleotide exchange factor, in cultured Aplysia neurons.

msec7‐1, a mammalian homologue of yeast sec7p, is known as a GDP/GTP exchange factor (GEF) for the ADP ribosylation factor (ARF) family of small GTPases. Here, we report that msec7‐1 overexpression in cultured Aplysia neurons leads to an extensive neuritogenesis in a GEF activity‐dependent manner through the modulation of the actin cytoskeleton. Similarly, the overexpression of ARNO, another mammalin GEF, produces extensive neuritogenesis in Aplysia neurons. In addition, msec7‐1 overexpression increases the number of varicosities with an altered size and shape in a GEF activity‐dependent manner. The overexpression of msec7‐1 in pre‐synaptic sensory neurons co‐cultured with post‐synaptic target motor neurons leads to an increase in the amplitude of the excitatory post‐synaptic potential through its GEF activity. Our results demonstrate that msec7‐1 regulates neuritogenesis and synaptic transmission.