SUMMARY The mechanism behind the phenomenon of "ligand functional selectivity”, defined as the ability of different ligands to differentially activate distinct signaling pathways through a common receptor, is not understood for receptor tyrosine kinases. Here we seek to obtain such mechanistic information for EphA2, a transmembrane receptor tyrosine kinase that is critically important for human health. We will investigate EphA2 signaling responses to three ligands: dimeric ephrinA1-Fc, monomeric m-ephrinA1, and the engineered monomeric YSA peptide ligand using biochemical and biophysical approaches in the context of live cells. We have already discovered differences in the extracellular configuration of the EphA2/YSA, EphA2/m- ephrinA1 and EphA2/ephrinA1-Fc oligomers in live cells, and in Aim 1 we will investigate whether these differences are transmitted across the plasma membrane, leading to differences in the configuration of the EphA2 intracellular regions. In Aim 2, we will compare EphA2 signaling responses to the three ligands to test the hypothesis that differences in downstream signaling correlate with differential phosphorylation of some of the EphA2 tyrosines and/or different kinetics of EphA2 activation/inactivation. This work will advance the general understanding of receptor tyrosine kinase signal transduction, and inform the design of “pathway-biased” targeted drugs with improved specificity and safety profiles.