Biomedical subjects
Publications and source records attributed to Martin Lackmann.
There is an urgent need for high purity, single chain, fully functional Eph/ephrin membrane proteins. This report outlines the pTIg-BOS-Fc vector and purification approach resulting in rapid increased production of fully functional single chain extracellular proteins that were isolated with high purity and used in structure-function analysis and pre-clinical studies.
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 Eph receptor tyrosine kinases and their membrane-bound ephrin ligands form a unique cell-cell contact-mediated system for controlling cell localization and organization. Their high expression in a wide variety of human tumors indicates a role in tumor progression, and relatively low Eph and ephrin levels in normal tissues make these proteins potential targets for anticancer therapies. The monoclonal antibody IIIA4, previously used to isolate EphA3, binds with subnanomolar affinity to a conformation-specific epitope within the ephrin-binding domain that is closely adjacent to the "low-affinity" ephrin-A5 heterotetramerization site. We show that similar to ephrin-A5, preclustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton, and cell rounding. BIAcore analysis, immunoblot, and confocal microscopy of wild-type and mutant EphA3 with compromised ephrin-A5 or IIIA4-binding capacities indicate that IIIA4 binding triggers an EphA3 conformation which is permissive for the assembly of EphA3/ephrin-A5-type signaling clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc applied in combination initiate greatly enhanced EphA3 signaling. Radiometal conjugates of ephrin-A5 and IIIA4 retain their affinity, and in mouse xenografts localize to, and are internalized rapidly into EphA3-positive, human tumors. These findings show the biological importance of EphA3/ephrin-A5 interactions and that ephrin-A5 and IIIA4 have great potential as tumor targeting reagents.
Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2.ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E alpha-helix, the E-F loop, the underlying H beta-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes.
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Eph receptor tyrosine kinases (Ephs) and their membrane anchored ephrin ligands (ephrins) form an essential cell-cell communication system that directs the positioning, adhesion and migration of cells and cell layers during development. While less prominent in normal adult tissues, there is evidence that up-regulated expression and de-regulated function of Ephs and ephrins in a large variety of human cancers may promote a more aggressive and metastatic tumour phenotype. However, in contrast to other RTKs, Ephs do not act as classical proto-oncogenes and do not effect cell proliferation or differentiation. Mounting evidence suggests that Eph receptors, through de-regulated re-emergence of their mode of action in the embryo may direct cell movements and positioning during metastasis, invasion and tumour angiogenesis. This review discusses these and other emerging roles of Eph receptors during oncogenesis.
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.
Eph receptors and their cell membrane-bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5-coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph-Eph interactions.
The EphA3 receptor tyrosine kinase preferentially binds ephrin-A5, a member of the corresponding subfamily of membrane-associated ligands. Their interaction regulates critical cell communication functions in normal development and may play a role in neoplasia. Here we describe a random mutagenesis approach, which we employed to study the molecular determinants of the EphA3/ephrin-A5 recognition. Selection and functional characterization of EphA3 point mutants with impaired ephrin-A5 binding from a yeast expression library defined three EphA3 surface areas that are essential for the EphA3/ephrin-A5 interaction. Two of these map to regions identified previously in the crystal structure of the homologous EphB2-ephrin-B2 complex as potential ligand/receptor interfaces. In addition, we identify a third EphA3/ephrin-A5 interface that falls outside the structurally characterized interaction domains. Functional analysis of EphA3 mutants reveals that all three Eph/ephrin contact areas are essential for the assembly of signaling-competent, oligomeric receptor-ligand complexes.
Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.
Pro-survival Bcl-2-related proteins, critical regulators of apoptosis, contain a hydrophobic groove targeted for binding by the BH3 domain of the pro-apoptotic BH3-only proteins. The solution structure of the pro-survival protein Bcl-w, presented here, reveals that the binding groove is not freely accessible as predicted by previous structures of pro-survival Bcl-2-like molecules. Unexpectedly, the groove appears to be occluded by the C-terminal residues. Binding and kinetic data suggest that the C-terminal residues of Bcl-w and Bcl-x(L) modulate pro-survival activity by regulating ligand access to the groove. Binding of the BH3-only proteins, critical for cell death initiation, is likely to displace the hydrophobic C-terminal region of Bcl-w and Bcl-x(L). Moreover, Bcl-w does not act only by sequestering the BH3-only proteins. There fore, pro-survival Bcl-2-like molecules probably control the activation of downstream effectors by a mechanism that remains to be elucidated.
Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions, membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.
The Eph and ephrin system, consisting of fourteen Eph receptor tyrosine kinase proteins and nine ephrin membrane proteins in vertebrates, has been implicated in the regulation of many critical events during development. Binding of cell surface Eph and ephrin proteins results in bi-directional signals, which regulate the cytoskeletal, adhesive and motile properties of the interacting cells. Through these signals Eph and ephrin proteins are involved in early embryonic cell movements, which establish the germ layers, cell movements involved in formation of tissue boundaries and the pathfinding of axons. This review focuses on two vertebrate models, the zebrafish and mouse, in which experimental perturbation of Eph and/or ephrin expression in vivo have provided important insights into the role and functioning of the Eph/ephrin system.