Abstract

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.