Neural cell adhesion molecule NCAM modulates dopamine-related behavior by regulating dopamine D2 receptor internalization in mice (Mus musculus Linnaeus, 1758)

Abstract

Dopaminergic transmission plays a critical role in the regulation of locomotion, cognition, reward and emotional behavior, and endocrine secretion. Responses to dopamine are controlled by a family of G protein-coupled proteins, which are classified to D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R) subfamilies. Increased activity of D2 receptor signaling is believed to play an important role in the pathogenesis of schizophrenia, and most antipsychotic drugs exert their functions by blocking D2 receptors. The signaling strength mediated by D2 receptor is extensively regulated by multiple processes, and endocytosis is a major mechanism of D2 receptor signal attenuation. However, the specific molecular mechanisms which modulate D2 receptor endocytosis have remained poorly understood. Neural cell adhesion molecule (NCAM), belonging to immunoglobulin superfamily, is a glycoprotein highly expressed and first discovered in the nervous system. Increasing evidence indicates that NCAM is related to psychiatric disorders, such as schizophrenia and bipolar disorders. Since both NCAM and D2 receptor are associated with psychiatric disorders, we become interested in a potential functional relationship between NCAM and D2 receptor. Here, we identified NCAM as a novel binding partner to D2 receptor. Co-immunoprecipitation revealed that NCAM could form complex with D2 receptor in physiological context, furthermore, the NCAM/D2R interaction was enhanced upon dopamine stimulation. Direct interaction was determined by pull down assay with recombinant NCAM and D2 receptor. Moreover, we clarified that the NCAM/D2R interaction was mediated via a short sequence stretch in the third intracellular loop of D2 receptor and the membrane-proximal part of the NCAM intracellular domain. To investigate the functional effect of NCAM/D2R interaction, subcellular distribution of D2 receptor in NCAM deficient mouse brains was analyzed. NCAM deficiency in mice led to increased D2 receptors at the plasma membrane and reduced D2 receptors in endosomes and lysosomes with unaltered total D2 receptor expression, indicating NCAM might be involved in the internalization process of D2 receptor. In vitro internalization assays and cell surface biotinylation experiment confirmed that NCAM promoted D2 receptor internalization/degradation and reduced cell surface localization of D2 receptor after dopamine stimulation. Furthermore, by in vivo pharmacological approach we could show that NCAM deficiency in mice resulted in excessive D2R-mediated signaling and exaggerated activity of dopamine-related behavior, which probably resulted from impaired internalization and increased levels of D2 receptors at the cell surface. These results demonstrate that, besides its classical function in cell adhesion, NCAM is involved in regulating the trafficking of neurotransmitter D2 receptor and receptor-related behaviors, thus implicating NCAM as an unexpected modulator for dopaminergic system and a potential pharmacological target for dopamine-related neurological and psychiatric disorders.