The guidance of axons to their targets during embryonic development is an intricate process requiring remarkable accuracy. The Eph family of receptor tyrosine kinases, and their ligands, the ephrins, proscribe many of these early axon guidance events. It is our hypothesis that oligomerization of these molecules controls both their signaling and their posttranslational modification, and in doing so confers upon them the accuracy and specificity of function necessary to shape the nervous system.
Aim 1 will determine the mechanism of ephrin B2 oligomerization, and its importance for receptor activation. Analytical ultracentrifugation will be used to determine whether ephrin B2 undergoes homophilic binding reactions. Further, an ephrin B2 binding protein, ABP, will be assayed for its ability to oligomerize ephrin B2. We will also use our newly developed system, fusing ephrin B2 to the lac repressor, to produce dimeric and tetrameric forms of the ephrin, which will be directly compared for their capacity to activate receptors.
Aim 2 and aim 3 will determine whether oligomerization of ephrin B2 drives its phosphorylation and degradation, respectively. The phosphorylation state of ephrin B2 will be measured after being clustered by mechanisms independent of the receptors. Ephrin B2 undergoes proteolysis in chick embryo fibroblasts. The proteolysis appears to be dependent upon clustering. We will determine whether dimers or tetramers undergo proteolysis. We will also use mutagenesis of ephrin B2 to determine which sequences are required for proteolysis.
The fourth aim will determine the importance of the biochemical interactions elucidated in the first three aims to the regulation of axon guidance. Derivatives of ephrin B2, designed to be resistant to posttranslation modifications, will be compared for their capacity to repel motor axons. In vitro studies will use the stripe assay, which tests the preference of an axon to extend over alternating stripes, containing, or lacking the appropriate chemorepellant. In vivo studies, will use the assay we have established to observe any deviations from normal of the motor nerve pathways, following ectopic expression of the ephrin B2 derivative from a retroviral vector in chick embryos. Through these comparative studies of ephrin function we will determine the post translation mechanisms which regulate their axon guidance activity.
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