[P200]: Role of ephrin B1 reverse signaling in development of the chick neural tube

Bi-directional signaling between Eph receptor tyrosine kinases and their membrane-bound ephrin ligands is required for the regulation of numerous physiological processes including neuronal differentiation and axon guidance. Though it is well established that ephrins possess Eph-receptor independent signaling activities, the signaling pathways that they employ remain largely unknown. Our central hypothesis is that the highly conserved regions of the cytoplasmic domain of transmembrane Bclass ephrins are required to induce a signalling event. Notably, the most well conserved region between all three B-class ephrins is found within the carboxy-terminal 33 amino acid cytoplasmic tail. This region contains five conserved tyrosine residues that when phosphorylated may recruit phospho-tyrosine binding proteins. In addition, there is a short polyproline stretch, as well as a PDZ-binding motif at the extreme carboxy-terminus. To directly investigate the function of the highly conserved cytosolic tail in B-class ephrin reverse signalling, we have constructed a number of ephrin B1 deletion mutants in this region and are currently testing their function both in vitro and in vivo – via virus – and electroporation-mediated gene transfer in NIH3T3 cells and the developing chick neural tube respectively. Intriguingly, in vitro functional analysis of wildtype protein has led to the serendipitous observation that ephrin B1 undergoes proteolytic processing via a gamma-secretase-like cleavage to produce a signalling competent fragment(s). We have therefore also constructed and are similarly testing a construct that lacks the majority of the extracellular domain of ephrin B1 but retains the entire transmembrane and intracellular domains. These experiments will reveal novel insight into the function of the cytosolic regions of ephrin B1 in the development of the chick neural tube.