The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse

Abstract

Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264).

Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

“…These residues are also part of a domain required for NCAM binding to heparin sulfate proteoglycans (HSPGs) 39 . In contrast, another Ig2 residue (NCAM R192) is not within the EphA3 binding site 38 . To assess the contribution of residues in NCAM Ig2 to neurocan binding, lysates of HEK293T cells expressing wild type (WT) NCAM or NCAM mutants (R156E, K162D, and R192E) were incubated with full-length neurocan, and the amount of neurocan co-immunoprecipitating with NCAM was analyzed by immunoblotting.…”

“…NCAM contains a heparin sulfate proteoglycan (HSPG) binding site in its Ig2 domain 37 , and residues within this proteoglycan binding domain (R156 and K162) are required for EphA3 binding 38 . Because the CSPG neurocan and HSPGs are extensively modified by GAG chain addition, it was hypothesized that neurocan engages the NCAM extracellular region (NCAM-EC) within the Ig2 proteoglycan binding domain.…”

“…Neurocan contains a carboxyl terminal sushi domain, a structure known to mediate protein-protein interactions. NCAM binds EphA3 at its cysteine-rich-domain (CRD), which also contains a sushi domain 38 . The Ig family member, L1-CAM, interacts with the sushi domain of neurocan 40 .…”

“…A complementary charged interface between the extracellular NCAM Ig2 domain and the EphA3 CRD mediates the binding and clustering of NCAM and EphA3 in the neuronal membrane. Amino acids R156 and K162 in the NCAM Ig2 domain were identified by molecular modeling and determined to be important residues for NCAM binding to EphA3 38 . These residues are also part of a domain required for NCAM binding to heparin sulfate proteoglycans (HSPGs) 39 .…”

ADP-ribosylation factor–like 4C binding to filamin-A modulates filopodium formation and cell migration

“…Confocal images of filopodia were obtained with a Zeiss LSM880 Confocal Upright Microscope with Airyscan with Carl Zeiss objective alpha Plan-Apochromat 100×/1.46 oil DIC VIS-IR and 1.8× optical zoom using Zeiss Zen software according to the manufacturer’s instructions. Zen digital imaging software (Zeiss) provides both microscopic image acquisition and processing as well as image data analysis and interpretation as evidenced in other published studies ( Merriman et al , 2016 ; Sullivan et al , 2016 ; Yap et al , 2016 ). Only Arl4C/FLNa, Arl4C/F-actin, Arl4C/fascin, or Arl4C/Cdc42-EGFP double-positive cells were imaged for analysis.…”