Eph receptor seeding mechanism. Detailed analysis of the mechanistic aspects of the
dimerizer-inducible Eph receptor clustering system has revealed that in contrast to an ephrinB2 stimulation, a nucleation or seeding effect is absent (this study and [104]). Seeding of Eph receptors was shown to be mediated by the extracellular receptor part [72,91,104], by contrast, dimerizer-induced clustering is induced by non-covalent crosslinking at the intracellular receptor part. This fundamental difference in the way clustering is provoked for Eph receptors can explain the absence of a seeding mechanism for dimerizer-induced clustering. Structural biologists claim that ephrin binding does not cause structural rearrangements on the intracellular receptor part but that conformational changes remain strictly limited to extracellular domains (personal communication with Elena Seiradake and [106]). In consequence, intra- and extracellular parts of Eph receptors are structurally most likely mutually inert to conformational changes. A clustering seed, originating from the intracellular part of the receptor, would thereby not be mediated to the outside structural part of the receptor and vice versa. By contrast, ephrin engagement as extracellular clustering trigger causes Eph receptors to snap in register to produce extended 1-dimensional signaling arrays or 2-dimensional platform-like structures [72,79,89,332]. For dimerizer-induced clustering this trigger is obviously missing, signaling array-like structures are, however, produced by distinct dimerizer-induced clustering (Fig. 4.1C). This lack of uncontrolled seeding ensures the distinctness of cluster size distributions as observed for the FKBP-system and shown by homo-FRET imaging and blue-native PAGE.
Overall, this difference in the mode of ephrin-induced versus dimerizer-induced cluster formation raises the question if thereby induced spatial signaling entities may differ in their cluster architecture influencing downstream signaling events. Cluster architecture can be categorized into an extracellular steric seeding platform consisting of Eph/ephrin ectodomain complexes (Fig 4.1B extracellular cluster entity) and an intracellular steric platform (Fig. 4.1B, intracellular cluster entity).
Signaling via the intracellular signaling platform. Intracellular kinase activation is the major consequence of Eph clustering and most important for biological functions in vivo [28,31]. However, receptor clustering may also provide a kinase-independent, sensitive intracellular structural configuration, which is sufficient or in addition to receptor autophosphorylation necessary for docking of signaling adaptors. Interestingly, the well- studied adaptor protein ephexin1 binds to EphA4 receptors (intersectin in the case of EphB receptors) in a constitutive manner [122,127,129], and its activation through phosphorylation is controlled by Eph clustering [113]. This modification is also mediated by kinase-active eeEphA4, suggesting that this is a process that happens largely independent of the regulation of EphA4 kinase activity. It was suggested that ephexin1 is recruited into higher-order
109 clusters of EphA4 along with Src family kinases and possibly other adaptor proteins. This then leads to the activation of Src kinase and subsequent ephexin1 phosphorylation by Src. My study reports, that dimerizer-induced clustering is sufficient to activate wtEph and N- terminally truncated Eph receptors and induce physiological signaling responses in heterologous cells and neurons. In light of the mechanistic aspects of the dimerizer-induced clustering system, it is highly unlikely that dimerizer-induced clustering forces the cytoplasmic domains by chance into an ordered signaling array that resembles the ephrin- induced physiological situation. Therefore a defined intracellular cluster configuration that might arrange specific interfaces for adaptor proteins is lacking and still, physiological Eph signaling responses are produced. Cell contraction responses are mainly mediated through the RhoA/ROCK/LIM kinase pathway through ephexin1 or intersectin to ultimately produce actin rearrangements [122,127,129]. Thus I conclude that dimerizer-induced clustering is sufficient to activate this pathway irrespective of producing a specific intracellular inter-receptor domain configuration [80]. Furthermore, recruitment of additional signaling components might either not be necessary to mediate kinase-dependent signaling responses or arbitrarily configured clusters are also sufficient for recruitment, e.g. through an unspecific co-clustering process.
Cell-surface signaling via the extracellular steric seeding platform. The extracellular cluster entity consists of multiple Eph/ephrin high-affinity interactions, which presumably potentiate along with growing cluster sizes to mediate strong adhesion to the opposing cell. This is, however, only the case in cell co-culture scenarios, where the cognate ligand is membrane-bound to the opposing cell. Dimer EphB2/ephrinB2 binding affinities are strong and dissociation constants (Kd) range in a sub-nanomolar order of magnitude [70,96]. The idea of extracellular strong and passive adhesive forces between cells can therefore be easily appreciated. This adhesive component of kinase-independent Eph function may be solely attributed to the extracellular steric seeding platform and reflects a pure passive mode of signaling mechanism. By contrast, kinase activity leading to trans-endocytosis of both receptor and ligand was shown to turn initial strong adhesion into contact-repulsion [237,238] involving an active kinase-dependent signaling process.
In this respect, both the FKBP-system and clustering with soluble ligand obviously fail to incorporate this kinase-independent mode of signaling through passive Eph/ephrin binding mediated adhesion. However, a secondary kinase-independent function may underlie the extracellular steric seeding platform. It may be able to evoke a signaling propensity, referred to as cell-surface signaling. The extracellular cluster entity may unspecifically co-cluster or specifically incorporate alien membrane proteins, i.e. produce cluster inclusions of alien (trans)membrane proteins, and thereby activate them.
Indeed, in this study, ephrinB2-Fc-induced clustering led to co-precipitation of an unidentified 50-55 kDa trans(membrane) protein. (Trans)membrane interacting partners in cis have been reported to be matrix metalloproteinases, γ-secretases, NMDA receptors [184,186,259], and
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other, so far unknown, specific or unspecific Eph cluster inclusions of alien (trans)membrane proteins are suspected. ADAM10 recruitment was demonstrated to correlate with cluster sizes produced by mechanical restriction of EphA2 [294], highlighting a possible kinase- independent role of cell surface signaling. In this respect my focus was not set on identifying the co-precipitated protein nor the mode of interaction but to find differences in ephrinB2-Fc induced versus dimerizer-induced clustering and thereby learn more about the molecular basis of receptor functioning. Interestingly, dimerizer-induced clustering failed to co-precipitate the unidentified protein. This difference in producing a competent cluster configuration for co- clustering of the alien (trans)membrane protein between ephrinB2-Fc induced and dimerizer- induced clustering may rely on the fact, that dimerizer-induced clustering is initiated intracellularly. Hence, this mode of clustering then fails to produce an extracellular steric seeding platform competent for recruiting cell surface, trans(membrane) signaling partners or effectors and furthermore accentuates an important mechanistic difference between ephringB2-Fc and dimerizer-induced clustering. Dimerizer-induced clustering most likely does not produce an extracellular steric configuration.