Plexin-B3 suppresses excitatory and promotes inhibitory synapse formation in rat hippocampal neurons.

Molecular mechanisms underlying synaptogenesis and synaptic plasticity have become one of the main topics in neurobiology. Increasing evidence suggests that axon guidance molecules including semaphorins and plexins participate in synapse formation and elimination. Although class B plexins are widely expressed in the brain, their role in the nervous system remains poorly characterized. We previously identified that B-plexins modulate microtubule dynamics and through this impact dendrite growth in rat hippocampal neurons. Here, we demonstrate that Plexin-B2 and Plexin-B3 are present in dendrites, but do not localize in synapses. We find that overexpression of all B-plexins leads to decreased volume of excitatory synapses, and at the same time Plexin-B1 and Plexin-B3 promote inhibitory synapse assembly. Plexin-B3 mutants revealed that these processes use different downstream pathways. While elimination of excitatory synapses is the result of Plexin-B3 binding to microtubule end binding proteins EB1 and EB3, the increase in inhibitory synapses is mediated by regulation of Ras and Rho GTPases. Overall, our findings demonstrate that Plexin-B3 contributes to regulating synapse formation.

B-plexins control microtubule dynamics and dendrite morphology of hippocampal neurons.

Semaphorins and their receptors plexins are implicated in various processes in the nervous system, but how B-plexins regulate the growth of dendrites remains poorly characterized. We had previously observed that Plexin-B1 and B3 interact with microtubule end-binding proteins (EBs) that are central adapters at growing microtubule tips, and this interaction is involved in neurite growth. Therefore, we hypothesized that plexins regulate microtubule dynamics and through that also dendritogenesis. The role of all three B-plexins was systematically examined in these processes. B-plexins and their ligand Semaphorin-4D influence the dynamics of microtubule tips both EB-dependently and independendently. EB3 as well as Plexin-B1, B2 and B3 turned out to have a significant role in the development of dendritic arbor of rat hippocampal neurons. Our results clearly indicate that semaphorin-plexin-EB pathway is one molecular mechanism how extracellular guidance cues are translated into intracellular mechanics. Taken together, Semaphorin-4D and B-plexins modulate the dynamic behavior of microtubule tips, and are therefore important in neurite growth.

Plexin-B3 interacts with EB-family proteins through a conserved motif.

BACKGROUND: Plexins are transmembrane receptors that are highly expressed in the central nervous system. They participate in the patterning of neural connections and regulation of cell adhesion and motility in many cell types. The aim of this study was to characterize novel protein-protein interactions of plexin-B3 intracellular portion. METHODS: To identify new interactors of plexin-B3 yeast two-hybrid screen was performed. We used GST pull-down and co-immunoprecipitation to verify those results. Deletion mutants were used to map the interacting regions. The physiological relevance of this interaction was assessed with neurite outgrowth assay in Neuro2A cell line. RESULTS: We show that the N-terminal segment of intracellular domain of plexin-B3 interacts with microtubule plus end-binding proteins EB1, EB2 and EB3. The corresponding region in human plexin-A2, B1 and B3 contains the conserved EB-binding motif SxIP and these plexins also associate with EBs indicating the specificity of plexin-EB binding. As to the EB proteins, their N-terminal microtubule-binding domain is dispensable for plexin interaction. Plexin-EB interaction is involved in neurite growth as the synthetic peptide corresponding to the EB-binding region of plexin-B1 increases significantly the number of neurite tips in Neuro2A cells. CONCLUSIONS: Microtubule end-binding proteins EB1, EB2 and EB3 interact with plexin-A2, B1 and B3 through a conserved EB-binding motif, which is located in their intracellular domain N-terminal segment. GENERAL SIGNIFICANCE: The observed interaction between plexin intracellular domain and EBs suggests a novel function for plexins in regulating EB-mediated changes in microtubule dynamics and neurite growth.

Identification and characterization of mouse plexin B3 promoter.

Plexin B3 is a neurally expressed transmembrane receptor protein participating in growth cone remodelling and synaptic plasticity. Despite their biological importance no plexin promoter has been characterized so far. Regulation of mouse plexin B3 transcription was investigated using in silico analysis of promoter and inititation area, 5'RACE, reporter-gene assays, gelshift and co-expression experiments. As a result we have described a novel 5' exon and show that a 234 basepair region upstream of it exhibits promoter activity. Further analysis indicated that this region contains predicted binding sites for myeloid zinc finger protein 1 (MZF-1) and neurogenin 3 (Ngn3). Oligonucleotides corresponding to the recognition sequences of these factors produced a specific mobility shift in EMSA. Expression of the reporter gene attached to the 234 region was increased 2-fold by Ngn3 and reduced twice in response to MZF-1 over-expression. These results help to better comprehend mechanisms used for plexin B3 transcriptional regulation.