Role of EphA1/A2 domain-domain and -membrane interactions for receptor specificity and Blood Brain Barrier dysfunction

Ligand activation of Eph receptors plays a decisive role in cell migration during blood vessel formation and neuronal axon guidance. Eph receptors can also signal independently of ligands, whereby the migration- and adhesion-repulsive signaling shifts to a pro-migratory stimulus that contributes to metastasis and drug

resistance in various cancers. Recent studies have substantiated the role of Eph receptors in the dysfunction of the blood brain barrier (BBB) during ischemic stroke, invasion of pathogenic organisms and the early stages of Alzheimer’s Disease (AD) and other neurodegenerative diseases. Yet, the molecular mechanisms behind

Eph receptor function remain poorly understood. Eph signaling depends on a variety of inter-molecular and intra-molecular Eph-Eph interactions which involve portions of the protein structure (domains) but also the cellular membrane. The overall project seeks to identify key residues in sets of interactions, which are not yet

well characterized, but are likely to be key to the different functional states of the overall protein interactions. Ephs are unique within the superfamily of transmembrane receptor tyrosine kinases due to their C-terminal 5- helix folded domain, part of the SAM (sterile alpha motif) adaptor protein family. We and others’ have reported

a novel role of the SAM domain to auto-inhibit EphA2 kinase activity. In preliminary experiments for this proposal, we discovered that mutations in the SAM domain functionally mimic its complete deletion and can abolish EphA2 autoinhibition. However, the molecular details of how the SAM domain inhibits the kinase

domain are still missing and will be investigated in Aim 1. EphA2 is cleaved at the cell surface by Membrane- type I matrix metalloproteinase and γ-secretase, key proteins for AD. However, the structure and function of these intracellular and extracellular-transmembrane receptor cleavage products are poorly understood. We will

characterize a protein construct encompassing the intracellular region (ICR), which consists of the JM region, the Kinase Domain (KD) and the SAM domain and another with the two membrane- proximal extracellular FibroNectin III domains (FN1&2), the transmembrane (TM), the juxtamembrane (JM) region. In aim 2, we will

use these EphA2 fragments to investigate the interaction between Eph domains and with the membrane. This knowledge is crucial for understanding the hierarchical organization of these regulatory interactions. The studies will be extended to the EphA1, EphA4 and EphB2 receptors, delineating how specific differences in

domain-domain contacts relate to different levels of kinase activity between the different Ephs. In addition, the proximity of the Eph receptor Fibronectin domains to the membrane is noteworthy: Our preliminary data for Aim 3 support the interaction between these domains and Aβ, a key peptide in AD. The structural insights we

pursue are essential for the development of diagnostic and therapeutic agents targeting neuronal and vascular diseases, including the breakdown of the blood brain barrier in neurodegenerative diseases.