Identification of novel cytosolic binding partners of the neural cell adhesion molecule NCAM and functional analysis of these interactions

Abstract

The neural cell adhesion molecule (NCAM) plays an important role during brain development and in adult brain. NCAM functions through interactions with several proteins leading to intracellular signal transduction pathways ultimately causing cellular proliferation, differentiation, migration, survival, and neuritogenesis. This thesis aimed for the identification of novel, yet unknown intracellular interaction partners of NCAM to further understand the mechanisms underlying NCAM’s role in the brain.<br /> Purified intracellular domains of human NCAM180 or NCAM140 were applied onto a protein macroarray containing 24000 expression clones of human fetal brain. Using this approach, several novel potential interaction partners were detected, including ubiquitin carboxyl-terminal hydrolase isozyme L1, ubiquitin-fold modifier-conjugating enzyme 1, and kinesin light chain 1 (KLC1). KLC1 is part of kinesin-1, a motor protein that transports cargoes towards the plus end of microtubules in axons and dendrites. As the transport mechanism of NCAM in neurons is still unknown, the potential role of kinesin-1 in NCAM trafficking was specifically interesting and analyzed in detail herein.<br /> The interaction of NCAM and KLC1 was verified in mouse brain tissue by co-immunoprecipitation. Co-localization studies in Chinese Hamster Ovary (CHO) cells overexpressing NCAM and kinesin-1 and in primary hippocampal neurons revealed an overlap of NCAM with subunits of kinesin-1.<br /> Functional studies showed that significantly more NCAM was delivered to the cell surface in NCAM and kinesin-1 overexpressing CHO cells. This effect was inhibited by excess of free full-length intracellular domain of NCAM as well as by several shorter peptides thereof. This showed that the intracellular domain of NCAM is required for the transport of NCAM to the cell surface. Further studies were carried out in primary cortical neurons. Whereas the kinesin-1 dependent transport of NCAM seemed to be mediated constitutively in CHO cells, the amount of cell surface NCAM significantly increased only after antibody-stimulated NCAM endocytosis in primary cortical neurons. In agreement, co-localization of internalized NCAM and KLC1 was observed in these neurons.<br /> Finally, an 8 amino acid sequence within the intracellular domain of NCAM was identified in an ELISA to be sufficient to directly interact with KLC1. The KLC1-binding region within NCAM overlaps with the domain responsible for binding to p21-activated kinase 1 (PAK1) which was shown to compete with KLC1 for binding to NCAM in a pull-down assay. This competition may provide a regulatory mechanism for the interaction between NCAM and KLC1 and could potentially be involved in the detachment of NCAM from KLC1 after delivery to the cell surface.<br /> Knowledge of the exact transport mechanism of NCAM will contribute to an advanced understanding of the underlying mechanisms of its functions during brain development and in adult brain.