| A number of molecules have been identified as guidance cues for migrating cells and neuronal processes within the last fifteen years. Cellular responses to these cues result in the rearrangement of the cytoskeletal network or the regulation of cell adhesion to facilitate migration. However, the mechanisms that allow guidance cues to communicate with the components of cell motility remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in directing cell and axon migration during neural development. Additionally, Eph receptors and ephrins have been hypothesized to participate in bidirectional signaling such that, upon receptor and ligand binding, a "forward" signal is transduced into the receptor-bearing cell while a "reverse" signal is sent into the ligand-bearing cell. Evidence from both genetic and biochemical studies suggest that the ephrin-B ligands may signal though their cytoplasmic domain. However, the mechanism for this reverse signaling is not clear.; In order to dissect the pathway downstream of ephrin-B reverse signaling, a yeast two-hybrid screen was performed to identify proteins that interacted with the cytoplasmic domain of ephrin-B ligands. Two ephrin-B binding proteins were identified: filamin, an actin filament crosslinker, and PDZ-RGS3, a novel protein. The studies presented here characterize the physical interactions of these proteins with ephrin-B molecules and their expression patterns in developing organisms. These studies provide insight into possible pathways for ephrin-B reverse signaling. Further work with PDZ-RGS3 in whole embryo assays and in cerebellar granule cells examine the functional role of the PDZ-RGS3/ephrin-B interaction. We find that both PDZ and RGS domains of PDZ-RGS3 are necessary to mediate ephrin-B induced de-adhesion in Xenopus embryos. In addition, ephrin-B reverse signaling stimulated by soluble EphB receptor can selectively inhibit granule cell chemoattraction to the chemokine SDF-1 in vitro and this activity is mediated by PDZ-RGS3. This work provides evidence which links reverse signaling to cellular guidance, reveals a mechanism for ephrin-B reverse signaling through PDZ-RGS3 by regulating heterotrimeric G protein coupled receptor signaling, demonstrates a mechanism for selective regulation of responsiveness to neuronal guidance cues, and proposes possible roles for ephrin-B molecules in cerebellar development. |
