Project Summary Signal transduction in development and disease (PI: Rohatgi) The goals of my research program are to uncover new regulatory mechanisms in cell-cell communication pathways, to understand how these mechanisms are damaged in disease states, and to devise new strategies to repair their function. Over the last 4.5 years, funding from the NIGMS has supported 23 publications across four different research areas in my laboratory: Hedgehog (Hh) signaling, WNT signaling, drug resistance mechanisms and intrinsically disordered proteins. Trainees involved in MIRA-supported research have won competitive fellowships (including a K99/R00 award from the NIGMS) and obtained independent group leader positions in both academia and industry. The next project period will tackle major unsolved problems in the vertebrate Hh and WNT signaling systems, two iconic cell-cell communication pathways that coordinate the construction of tissues during development and their subsequent maintenance throughout adult life. Despite the importance of these pathways in human diseases ranging from birth defects to cancer and degenerative conditions, many steps in Hh and WNT signaling remain poorly understood at the biochemical and cell biological level. In the Hh pathway, our focus is on understanding how a signal is detected at the cell surface and transmitted across the plasma membrane to transcriptional effectors in the cytoplasm. These signaling steps in the vertebrate Hh pathway depend on primary cilia, antenna-like organelles that project from the surfaces of most cells and are implicated in human birth defect syndromes called “ciliopathies.” Major questions under investigation include (1) how Patched 1 (PTCH1), the receptor for Hh ligands, regulates the function of Smoothened (SMO), the protein that transmits the signal across the membrane, (2) how SMO is activated at primary cilia and (3) how SMO signals to the Glioblastoma (GLI) family of transcription factors. Our MIRA- supported work has led to a new paradigm in transmembrane signaling: the use of cholesterol accessibility in the ciliary membrane as a second messenger to communicate the signal between PTCH1 and SMO. Our focus in the WNT pathway is on the multi-protein β-catenin destruction complex that suppresses WNT signaling by promoting the degradation of β-catenin. Defects in this complex drive the vast majority of colorectal cancer, a disease with an increasing burden (especially amongst people <50 years of age) predicted to cause over 1 million deaths yearly by 2030. Our emphasis is on uncovering differences in the genetic and biochemical requirements for oncogenic (mutation-driven) and physiological (ligand-driven) WNT signaling, since any successful anti-WNT drug will have to distinguish between the two to achieve an acceptable therapeutic index. Our work is supported by long-term collaborations and embraces a broad range of techniques that span structural biology, lipid biochemistry, CRISPR/Cas9-base...