During normal development progenitor cells of many tissues undergo progressive restriction of pluripotency, epithelial-to-mesenchymal transition, proliferation, migration, and differentiation. Most, if not all, of these events involve modifications of cell-cell and cell-matrix adhesion, and abnormal modifications of these adhesion systems are often associated with the formation of tumors. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are frequently over-expressed in a wide variety of cancers, including breast, small-cell lung and gastrointestinal cancers, melanomas, and neuroblastomas. Using the Xenopus embryonic system, we have demonstrated that signaling mediated by the intracellular domain of ephrinB affects cell-cell adhesion, and that this activity can be modulated by interaction with an activated FGF receptor. This year, we and our collaborators presented evidence that ephrinB1 signaling alters the gastrulation movements of retinal stem cells and is necessary for these cells to move into the eye field. Moreover, cross-talk with the FGF receptor system can control the ephrinB1-guided morphogenetic movements of retinal progenitors. These results implicate cooperative signaling between FGF and ephrin signaling pathways in the early stages of retinal progenitor specification and movement. Furthermore, it suggests the possibility that Eph/ephrin signaling may be linked to oncogenesis through their role in development, including control of adhesion, and motility. Among the more exciting tasks at hand are defining which molecular interactions with the intracellular domains of Eph receptors and ligands have a physiological function, and determining the mechanisms by which Eph receptor and ligand signaling cascades are propagated and regulated. Toward this end, previous work from our laboratory has implicated the activated fibroblast growth factor receptor1 (FGFR1) as a regulator of a de-adhesion signal that results from over-expression of ephrinB1. This year, we report the isolation of Xenopus Grb4, an ephrinB1-interacting protein, and show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4 and the resulting mutants were expressed along with ephrinB1 and an activated FGF receptor in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the SH2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, while tyrosines 305 and 310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine phosphorylation-dependent event.
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