The Eph family of receptor tyrosine kinases are expressed in regulated patterns in the developing vertebrate brain. These receptors are thought to control guidance, outgrowth, or fasciculation of axons, however the mechanisms by which receptor activation promotes changes in axon behavior are ill-defined. In this proposal we plan to develop an in vitro system in which we can dissect the relationship between the biochemical consequences and the biological effects of Eph receptor activation in cortical neurons. We will begin by characterizing the biological outcome of Eph receptor activation in cortical neurons growing on a substrate of membranes laid down in stripes that alternate in the expression of the Eph receptor ligand ephrin-A1. This assay has been used in other studies to demonstrate that axons are repelled from regions of high ephrin expression. Once we have established this bioassay, we will focus on the effects of EphA4 receptor activation. We will isolate activation of this specific receptor by expressing its intracellular domain in the context of a chimera with the extracellular domain of the FMS receptor. Application of the FMS ligand, M-CSF, should activate only the chimera independent of other Eph receptors, showing only the biochemical and biological outcomes downstream of this receptor. Finally, by analyzing what changes in the outcomes occur with mutations of the chimera that alter coupling to signal transduction pathways, we will have evidence for the signaling mechanisms by which the Eph receptors effects their biological functions in neurons.
