Structural regulation of megalin recycling in the proximal tubule

Abstract This K08 Career Development Proposal details the candidate’s research on the structural regulation of megalin recycling in the kidney proximal tubule (PT) that will be conducted during a 5-year mentored award period. With the guidance of his co-mentors at Columbia University, Dr. Shapiro and Dr. Barasch, along with oversight from an experienced multi-institutional advisory committee, Dr. Beenken has designed a comprehensive plan of didactics and intensive laboratory research in cryo-electron microscopy (cryo-EM) and mass spectrometry. This training will enable him to differentiate himself from his mentors and develop a body of research that he can use to start an independent research laboratory as a physician-scientist. His ultimate goal is to translate the understanding of structural mechanisms of megalin recycling to new treatments for proteinuric disease. Megalin-dependent endocytosis in the PT enables protein capture that protects the kidney tubules from damage due to proteinuria. Megalin must recycle between cell surface and endosomes to fulfill its endocytic function, but the structural mechanisms that underlie this recycling are poorly understood. Dr. Beenken has published cryo-EM structures of megalin at pH 7.5 and 5.2, revealing megalin’s structure at the cell surface and in the late endosome. These structures demonstrated that megalin undergoes extensive pH-dependent structural transitions to bind ligands from the urinary filtrate at the cell surface and then shed ligands in the acidic endosomes. Dr. Beenken’s work led him to hypothesize that megalin adopts unique structures in different endosomal compartments that are regulated by specific residues in megalin and depend on changes in megalin’s coordination of Ca2+ ions. In his proposed research project, Dr. Beenken will test his hypothesis by determining structures of megalin in different endosomal compartments including early and recycling endosomes (Aim 1), characterizing pH-sensitive megalin residues during trafficking (Aim 2), and determining the dependence of megalin recycling on Ca2+-coordination (Aim 3). Pursuing these research goals will lay the groundwork for Dr. Beenken’s career in the structural biology of megalin receptor recycling in the PT and enable him to become an independent investigator. Ultimately, he plans for his discoveries in the basic science of megalin recycling in the PT to translate to clinical interventions for proteinuric disease.