Project Summary/Abstract Many therapeutics for proliferative and neurodegenerative disorders, such as cancer and amyotrophic lateral sclerosis (ALS), aim to disrupt or inhibit downstream signaling pathways components such as the MAPK cascade. A critical barrier in therapeutic development against Receptor Tyrosine Kinases (RTKs) is that oftentimes signaling pathways are interconnected, and this may lead to off-target side-effects. One method to mitigate these challenges is to determine the mechanism of activation of membrane receptors that actively play a role in abnormal cellular functions. RTKs, such as Erythropoietin-producing hepatoma (Eph) receptor 4, have been of particular interest as potential drug targets. However, there is a lack of structural information on EphA4 that provides insight into receptor activation. In this work, we aim to determine key structural differences during activation of dimeric EphA4 as well investigate the activity of loss-of-function EphA4 mutants using complementary fluorescence-based and structural studies to validate our findings. In aim one, we propose to elucidate critical structural differences resulting in wild type EphA4 activation using cryo-electron microscopy (cryo-EM). In aim 2, we propose to characterize aberrant activity and oligomerization of known EphA4 cancer mutants through fluorescence-based studies, biophysical, and biochemical assays. The results of these aims will contribute significantly to our underlying understanding of RTKs and will aid in efforts to develop EphA4 structure-based drug designs for proliferative and neurodegenerative disorders.