The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.

The myelin‐derived proteins Nogo, MAG and OMgp limit axonal regeneration after injury of the spinal cord and brain. These cell‐surface proteins signal through multi‐subunit neuronal receptors that contain a common ligand‐binding glycosylphosphatidylinositol‐anchored subunit termed the Nogo‐66 receptor (NgR). By deletion analysis, we show that the binding of soluble fragments of Nogo, MAG and NgR to cell‐surface NgR requires the entire leucine‐rich repeat (LRR) region of NgR, but not other portions of the protein. Despite sharing extensive sequence similarity with NgR, two related proteins, NgR2 and NgR3, which we have identified, do not bind Nogo, MAG, OMgp or NgR. To investigate NgR specificity and multi‐ligand binding, we determined the crystal structure of the biologically active ligand‐binding soluble ectodomain of NgR. The molecule is banana shaped with elongation and curvature arising from eight LRRs flanked by an N‐terminal cap and a small C‐terminal subdomain. The NgR structure analysis, as well as a comparison of NgR surface residues not conserved in NgR2 and NgR3, identifies potential protein interaction sites important in the assembly of a functional signaling complex.

SUMMARY The endothelial specific receptor tyrosine kinase Tie2, and the orphan receptor tyrosine kinase, Tie1, are essential for endothelial cell proliferation, migration, and survival during angiogenesis. Despite their considerable similarity, experiments with Tie1 or Tie2 deficient mice highlight distinct functions for these two receptors in vivo. However, Tie1 cooperates with Tie2 during Angiopoietin signaling, demonstrating a degree of functional overlap between the two receptor-systems. Tie2 is further unique among receptor tyrosine kinases with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists depending upon the local environment, while Angiopoietin-1 and -4, are constitutive agonists. To address the role of Tie1 in Angiopoietin-mediated Tie2 signaling, and determine the basis for the unique behavior of the individual Angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and Tie2 localization and association in the presence and absence of ligands. Here, we provide direct evidence for Tie1-Tie2 complex formation on the endothelial cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by Angiopoietin-1 and -2, thereby providing a discrete molecular mechanism for the observed variations in Angiopoietin function. Based upon the available data, we propose a unified model for Angiopoietin-induced Tie2 signaling which highlights the essential role of Tie1 in vascular homeostasis.