Mechanisms of Eph/ephrin mediated cell-cell communication

Eph receptors and their membrane associated ephrin ligands mediate
cell-cell repulsion to guide migrating cells and axons. A
peculiarity of this signaling system is that both receptors and
ligands can transduce signals into the cell resulting in a
bidirectional signaling mode. An important step of ephrinB ligand
‘reverse signaling’ is the regulated tyrosine phosphorylation of
the cytoplasmic domain, initiating docking sites for downstream
signaling adaptors. Moreover, ephrinB ligands can signal by
interactions with various PDZ-domain containing proteins. Using a
broad range of in vitro assays the presented work demonstrates that
upon binding to their cognate receptor, ephrinB ligands rapidly
activate Src family kinases (SFKs) which subsequently phosphorylate
ephrinB. Tyrosine phosphorylation appears to be a transient event
that is downregulated by the tyrosine phosphatase PTP-BL, which
interacts with ephrinB via one of its PDZ-domains. Studies on
PTP-BL and another multiple PDZ domain containing protein GRIP
(Glutamate Receptor Interacting Protein) revealed that PDZ
interactions with ephrinB are also regulated by EphB receptor
binding but, unlike tyrosine phosphorylation, these interactions
are long lasting. These findings led to postulate a ‘switch model’
for ephrinB reverse signaling: Tyrosine dependent signaling appears
to be a rapid and transient event which is later replaced by stable
PDZ-dependent signaling. EphB ‘forward’ signaling as well as
ephrinB ‘reverse’ signaling are important for axonal pathfinding
and cell migration during development. Prior to their repellent
effect on migrating cells and growth cones, Eph receptors form a
high affinity complex with their ligands at sites of cell-cell
contact. Therefore, mechanisms have to be in place that allow cells
to detach from each other permitting retraction and withdrawal. To
overcome this adhesive barrier, the ectodomain of ephrinA ligands
is cleaved by metalloproteinases and shed upon receptor binding.
Intrigued by the previous findings that activated ephrins cluster
in cells, we hypothesized that these Eph/ephrin clusters undergo
endocytosis. We developed immunofluorescence internalization and
co-culture assays to study clustering and endocytosis at cell-cell
contact sites. We established an experimental setup to perform fast
time lapse imaging studies of cells expressing different
fluorescently tagged proteins. Cell contact-induced ephrinB-EphB
complexes are rapidly endocytosed during the retraction of cells
and neuronal growth cones. Endocytosis occurs in a bidirectional
manner, leading to internalized complexes of full length receptor
and ligand, a yet rarely observed phenomenon. Signaling inactive
mutants of EphB receptors and ephrinB ligands lead to a strong
adhesion between cells. Endocytosis is sufficient to convert this
adhesion into the detachment of cells. Bidirectional endocytosis is
necessary to efficiently promote axon detachment during growth cone
collapse mediated by ephrinB ligands. On the cell biological level,
bidirectional endocytosis of two full length transmembrane (TM)
proteins is a new phenomenon. Moreover, these studies reveal a
novel mechanism of signal termination, de-adhesion and promotion of
cell repulsion after intercellular (trans) interaction between two
TM proteins.