New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization.

Antoni Parcerisas,Eduardo Soriano,Eliandre De Oliveira,Maria Antonia Odena,Lluís Pujadas,Miquel Bosch,Alba Ortega-Gascó,Fausto Ulloa,Marc Hernaiz-Llorens

doi:10.3390/ijms22147404

Abstract

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.

Highlights

Neuronal differentiation, and the establishment of cell polarity and synaptic connections, are crucial events for the development of the brain [1,2]
We explored the interactome of Neuronal cell adhesion molecule 2 (NCAM2) and revealed a significant role of this protein in the organization and dynamics of the cytoskeleton
The mass spectrometry approach showed that NCAM2 interacts with key cytoskeleton components and with a large number of other intracellular proteins

Summary

Introduction

The establishment of cell polarity and synaptic connections, are crucial events for the development of the brain [1,2]. These processes are tightly regulated by numerous factors, including cytoskeleton proteins, membrane receptors and elements of the extracellular matrix. The immunoglobulin superfamily of cell adhesion molecules (IgSF CAM) includes more than 50 different members in mammals [6]. Most IgSF CAMs present a single transmembrane domain with an intracellular tail, while other members are anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor [7]. The extracellular and the intracellular domains of IgSF CAMs interact with several proteins, ligands or modifiers, which determine their important roles during development [5,8,9]

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Conclusion