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Weinges, Stefan (2006): Molecular dissection of ephrinB reverse signaling. Dissertation, LMU München: Faculty of Biology
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Abstract

Synapses form when highly motile dendritic filopodia establish axonal contacts. When a synaptic contact is stabilized, it gives rise to the formation of a dendritic spine, which has recently been shown to involve a number of molecules that mostly regulate the actin cytoskeleton. Thus, it is not surprising that Eph receptor tyrosine kinases, as known regulators of signaling pathways involved in actin cytoskeleton remodeling, have been shown to be required for spine development and maintenance. The main characteristic of interactions of the Eph receptor with its membrane associated ephrin ligand is that they can propagate bidirectional signals. Both forward (downstream of Eph receptor) and reverse (downstream of ephrin ligand) signaling have been shown to play a role in mature synapses, where spine morphology changes are associated with synaptic plasticity. Thus, ephrinB reverse signaling might be as important for dendritic spine development as signaling pathways downstream of Eph receptors. Intrigued by this idea, we hypothesized that some of the spine morphology changes during plasticity might be regulated exclusively by ephrin reverse signaling pathways. Analyzing spine formation in cultures of dissociated hippocampal neurons, we demonstrated that stimulation of hippocampal neurons with EphB receptor bodies leads to increased spine maturation. Expression of a truncated form of ephrinB ligand, which is still able to activate EphB receptor but is unable to transduce intracellular signals, impairs spine morphology. To find new players of reverse signaling that are important in directing ephrin-mediated spine morphology, we performed a proteomic analysis of the phosphotyrosine dependent ephrin interactor Grb4 (Nck-2, Nck beta). We identified the signaling adaptor G protein-coupled receptor kinase-interacting protein (GIT)1 (Cat1) as well as the exchange factor for Rac βPIX (β-p21-activated protein kinase (PAK)-interacting exchange factor), also called RhoGEF7 or Cool-1, as novel Grb4 binding partners, which have both previously been shown to be required for spine formation. We show that Grb4 binds and recruits GIT1 to synapses downstream of activated ephrinB ligand. Interactions of Grb4 with ephrin or GIT1 are necessary for proper spine morphogenesis and synapse formation. We therefore provide evidence for an important role of ephrinB reverse signaling in spine formation and describe the ephrinB reverse signaling pathway involved in this process.