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The development of dendritic arbors is critical in neuronal circuit formation, as dendrites are the primary sites of synaptic input. Morphologically specialized dendritic protrusions called spines represent the main postsynaptic compartment for excitatory neurotransmission. Recently, we demonstrated that chicken acidic leucine-rich epidermal growth factor (EGF) -like domain-containing brain protein/neuroglycan C (CALEB/NGC), a neural member of the EGF family, mediates dendritic tree and spine complexity but that the signaling pathways in the respective processes differ. For a more detailed characterization of these signal transduction pathways, we performed a yeast two-hybrid screen to identify proteins that interact with CALEB/NGC. Our results show that B56beta, a regulatory subunit of protein phosphatase 2A, interacts with CALEB/NGC and inhibits CALEB/NGC-mediated dendritic branching but not spine formation. Binding of B56beta to CALEB/NGC was confirmed by several biochemical and immunocytochemical assays. Using affinity chromatography and mass spectrometry, we demonstrate that the whole protein phosphatase 2A trimer, including structural and catalytic subunits, binds to CALEB/NGC via B56beta. We show that CALEB/NGC induces the phosphorylation of Akt in dendrites. Previously described to interfere with Akt signaling, B56beta inhibits Akt phosphorylation and Akt-dependent dendritic branching but not Akt-independent spine formation induced by CALEB/NGC. Our results contribute to a better understanding of signaling specificity leading to neuronal process differentiation in sequential developmental events.

The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF-like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC-induced stimulation of dendritic branching. In contrast, CALEB/NGC-induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.

CALEB/NGC is a neural member of the epidermal growth factor protein family expressed in axon and synapse-rich areas of the nervous system and shown to be important for neurite formation. It can bind to the extracellular matrix proteins tenascin-R and tenascin-C. Here we show that CALEB/NGC interacts with the Golgi-associated protein PIST. PIST was originally described as an interaction partner of the small GTPase TC10 and was then found to be Golgi-associated by binding to syntaxin-6 and to be important for the transport of frizzled proteins and the cystic fibrosis transmembrane conductance regulator to the plasma membrane. In addition, PIST was demonstrated to be involved in autophagy and linked to processes of neurodegeneration. CALEB/NGC interacts with PIST in the yeast two-hybrid system. This interaction can be confirmed by co-immunoprecipitations and co-localization studies. The juxtamembrane cytoplasmic peptide segment of CALEB/NGC, highly conserved during evolution, mediates the binding to PIST. CALEB/NGC co-localizes with PIST in the Golgi apparatus of transfected COS7 cells and in Golgi-derived vesicles after brefeldin A or nocodazole treatment. Co-localization studies in primary hippocampal cells and analysis of Purkinje cells of colchicine-treated rats, serving as an in vivo model system to block microtubule-dependent transport processes, support the view that PIST is an interaction partner of CALEB/NGC and implicate that this interaction may play a role in the intracellular transport of CALEB/NGC.