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RTK signaling in general, but also Eph signaling in particular, is influenced by trafficking and processing prior and posterior to receptor activation. Upon ligand engagement, the amplitude and kinetics of signaling may be determined by a highly regulated endocytic process, which sorts activated receptors to degradation in lysosomes eventually leading to complete attenuation and termination of the initial signaling response (Fig. 1.6) [10,235].

Regulation of Eph internalization. Eph/ephrin complexes undergo internalization by

endocytosis upon cluster formation and activation [174]. During this process, Eph/ephrin complexes at cell-contact interfaces are rapidly removed from the cell surface by trans- endocytosis into both the Eph-expressing cell (forward endocytosis) and the ephrin- expressing cell (reverse endocytosis) to terminate adhesion and allowing for contact-mediated repulsion [236-239]. Interestingly, trans-endocytosed Eph/ephrin complexes are still associated to their original membrane domains leading to double-membrane-coated intracellular vesicle structures [237,240]. Furthermore, trans-endocytosed Eph/ephrin complexes persist in signaling suggesting that active signal transduction can be redirected into one or the other adjacent cell depending on the balance of endocytic processing [237,238]. To date, the underlying mechanism for this rather unusual process is not known.

Activity of the small GTPase Rac1 is required for local rearrangements of the actin cytoskeleton to cause membrane ruffling for initiation of endocytic processes [156,237].

Fig. 1.6 Eph/ephrin processing and endocytosis.

Schematic presentation of Eph/ephrin processing by proteases, Eph pre/post-activation trafficking and

surrounding signaling pathways as described in the main text. Arrows () denote a positive, (T)-

indicators a negative regulation on downstream targets. The location of the signaling proteins does not imply the involvement of a particular Eph/ephrin domain. The question marks indicate signaling connections that have not been conclusively assessed in Eph/ephrin signaling. Eph processing and trafficking may depend on the Eph/ephrin levels, degree of ligand/receptor clustering, and cellular context. Some of the mechanisms were derived from studies using soluble Fc fusion proteins and are not validated by cell-cell stimulation experiments.

27 EphB2/CTF2 peptide P P P P P P Src

B

A

A/B

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Recent work has implicated the Rac exchange factor Vav (Vav1-3) in Eph receptor forward endocytosis. In contrast to the related ephexin1, Vav2 has no preferences for EphA or EphB and binds through its single SH2 domain to activated, phosphorylated JM-tyrosines subsequently enhancing Rac activation and Eph endocytosis [241]. Vav2/3 double knockout mice develop axon guidance defects, presumably due to the absence of Eph-mediated axon growth cone collapse in response to ephrinA1 [241]. Similar to Vav2 for EphA4, other GEFs like TIAM1 play an important role for EphA8-dependent Rac1 activation and its internalization [242].

SHIP2 was discovered as a negative regulator of ligand-induced EphA2 endocytosis. It binds to EphA2 via a heterotypic SAM-SAM domain interaction [156]. SHIP2 dephosphorylates PIP3 (phosphatidylinositol 3,4,5-trisphosphate) thereby suppressing PI3K signaling, which in turn is required for enhancing Rac1 activation [243].

Eph endocytic pathways. While it appears that Rac1 signaling generally enhances

Eph/ephrin internalization [156,237], the exact role of the clathrin endocytosis machinery remains unclear [174]. To date, the only two studies linking Eph receptor internalization to the clathrin-dependent pathway show involvement of TIAM1 and synaptojanin1 [242,244]. Importantly, stimulation of cells with soluble Eph or ephrin fused to the Fc portion of human immunoglobulin likely activates endocytic pathways, which significantly differ from the ones induced by physiological cell-cell interactions. Indeed, a phospho-proteomic study has reported a different selection of targets and adaptors to be activated when soluble ephrin-Fc was used for stimulation [245].

In contradiction to a strictly clathrin-dependent mode of internalization, Ephs were also discovered to be concentrated in caveolae, and the EphB1 receptor to be associated with the protein caveolin-1 [246]. These few reports indicate that further work is required to elucidate the full molecular mechanism of Eph/ephrin endocytosis or identify other modes of internalization like caveolae or pinocytosis.

Eph trafficking. Vesicles loaded with receptor cargo undergo extensive sorting and

maturation to various endocytic compartments. The Rab GTPase proteins are highly compartmentalized in organelle membranes and together with their effectors coordinate these consecutive stages of transport, which comprise processes like vesicle formation, vesicle motility and tethering of vesicles to their target compartment [247]. Eph receptor trafficking prior to and post ligand-engagement and cluster formation has only been poorly addressed so far.

Rin1 (Ras/Rab interactor 1) is a GEF for Rab5, which is known to control the fusion of endocytic vesicles and early endosomes. Like Vav, it was found to bind to EphA4 via its SH2 domain and to become phosphorylated upon EphA4 activation, causing the EphA4-sorting in Rab5-positive compartments. In vivo, EphA4 and Rin1 control neuronal plasticity in opposite

29 ways, suggesting that Rin1 antagonizes EphA4 function through induction of an enhanced internalization response [248].

The Eph receptor’s final fate upon ephrin engagement and internalization was speculated to be degradation in lysosomes as observed for other RTKs as a mechanism to shut down signaling [10]. In fact, ligand binding induces cbl-dependent ubiquitinylation and EphB1 degradation through the lysosomal pathway, also marked by the late endosomal marker Rab7 identified in a proteomics approach and co-localization studies with lysosomal compartments [104,249,250]. However, these studies rely on a strong stimulation with soluble ephrin-Fc, which might not reflect the physiological situation of Eph stimulation response in vivo. It may be speculated that a rather differential trafficking response comprising recycling components aside from the crude lysosomal fate may also apply for Eph receptors, which e.g. only undergo mild clustering.

Ephs, as all other transmembrane receptors, are secreted from Golgi compartments to the plasma membrane, after translation and glycosylation [2]. For EphB2, in hippocampal neuronal cultures, a kinesin1-dependent anterograde transport mechanism to dendrites was observed involving the PDZ-adaptor protein GRIP1 [251] (Fig. 1.6).

Ephrin reverse endocytosis. The clathrin-dependent pathway has also been implicated in reverse endocytosis. GFP-tagged ephrinB1 co-localizes in clathrin-coated vesicles, positive for the early endosome marker EEA1 (early endosome antigen 1) after stimulation with CHO (Chinese hamster ovary) cells expressing EphB1 [252] (Fig. 1.6). As small GTPases are also activated downstream of B-class ephrins [209,253,254], GEFs might be good candidates for the regulation of reverse endocytosis.

EphrinB2 seems to be destined for degradation through the proteasomal pathway after stimulation with soluble EphB2-Fc. In Xenopus retina cultures, ephrinB2 degradation was inhibited by proteasome-specific inhibitors LnLL (N-acetyl-l-leucinyl-l-leucinal-l- norleucinal) and lactacystin [236].

Eph/ephrin cleavage. Cleavage of Ephs and ephrins by ADAM (A-Disintegrin-And-

Metalloprotease) family metalloproteases and γ-secretase proteases is an additional mechanism to terminate Eph/ephrin contact. EphrinA2 was identified to associate with ADAM10, which cleaves the ephrinA ectodomain thus facilitating contact-repulsion of axons [255].

In addition, ADAM10 was also proven to interact with EphA3 and cleave ephrinA2 in trans only after binding to EphA3 [256,257] (Fig. 1.6).

For ephrinBs, γ-secretase-dependent cleavage takes place in cis but not in trans [210,258]. Recently, EphB2 ectodomain release to the extracellular space was evidenced following cleavage after EphB2 residue 543 insensitive to metalloproteinase inhibitor GM6001 [186]. Here, EphB2 is in addition cleaved by a presenilin-dependent γ-secretase activity releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. Interestingly, cytosolic

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peptides produced by the combined metalloproteinase/γ-secretase processing of cell surface proteins can function in signal transduction and protein phosphorylation [186]. Inhibition of ephrinB2-induced EphB2 cleavage also reduces Eph-mediated axon growth cone collapse [259]. Moreover, the released EphB2 intracellular peptide was shown to be important for NMDAR-subunit phosphorylation in primary neuronal cultures [260]. In the case of EphA4, intracellular peptide enhances preferentially Rac signaling [261].