Molecular dissection of ephrinB reverse signaling

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.