BPS2026 – Discovering the structural basis of activation in the TAM receptor subfamily

Receptor tyrosine kinases (RTKs) play critical roles in cellular processes such as metabolism, cell proliferation, differentiation, migration, and apoptosis. RTKs are activated by ligands binding to extracellular domain receptors. Traditionally, bound ligands are thought to cause receptor dimerization and subsequent phosphorylation of tyrosine residues on the cytosolic domain, inducing downstream signaling cascades. TAM receptors (Tyro3, Axl, and Mer) are a unique RTK subfamily of interest with primary roles in cell cycle regulation and apoptosis. They are activated by two ligands: Gas6 and Protein S. Axl, which is involved in cancer, spermatogenesis and platelet function, has single-digit nanomolar binding affinity for Gas6, but how this interaction promotes activation remains poorly understood. Currently, the only known TAM receptor:ligand structure is a fragment of the Axl extracellular domain bound to truncated Gas6. Axl activation is thought to require Gas6 binding to the membrane lipid phosphatidylserine, suggesting the available structure may not represent an active complex. We hypothesize that there will be detectable structural changes when a TAM receptor:ligand complex is bound to lipid. My project aims to better understand Axl activation by analyzing full-length Gas6 interactions with Axl, with and without lipid, using structural methods. I have successfully expressed both the full length extracellular domain of Axl and Gas6 from insect and mammalian cell lines, respectively. Protein purification methods for both proteins have been optimized, enabling cryo-EM data collection. Data processing through CryoSPARC has yielded an initial partial 3D map of the Axl:Gas6 complex at 3.6 Å resolution. Future work will optimize grid conditions for the Axl:Gas6 complex, with and without lipid, for improved resolution and include further data collection and processing.