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.
