Mechanisms of Smoothened Activation in Hedgehog Signaling

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PROJECT SUMMARY/ABSTRACT MECHANISMS OF SMOOTHENED ACTIVATION IN HEDGEHOG SIGNALING. The Hedgehog (Hh) signaling pathway coordinates development in humans and most metazoans. In adult tissues, aberrant Hh pathway activation drives various cancers, including basal cell carcinoma of the skin and pediatric brain medulloblastoma. An unusual mechanism enables Hh signal transduction. In the basal state, the transporter- like molecule Patched1 (PTCH1) represses Smoothened (SMO), a seven-transmembrane protein. Hh inhibits PTCH1, which unleashes SMO activity causing downstream transcription. A leading mechanistic hypothesis is that cellular sterols activate SMO, but are sequestered by PTCH1 in the absence of Hh. Connecting the putative transport function of PTCH1 to SMO activation remains one of the central mysteries in understanding how the Hh signal is transduced across the membrane. A critical gap is how, and which, sterols directly influence SMO activation. This proposal will address these questions in two aims. The first aim focuses on which sterols activate SMO. Here, we will combine biochemistry, structural biology, and signaling studies to define which sterols can activate SMO and how they do so. In the second aim, we focus on how sterol binding to distinct sites on SMO influences Hh pathway activity. We will use X-ray crystallography, cryo-electron microscopy, and NMR spectroscopy to generate an integrated high-resolution perspective on where sterols bind to SMO, and how they influence SMO activity. These studies will provide a mechanistic picture on how the Hh signal is transduced across the membrane. Such insights may enable the design of SMO inhibitors less susceptible to tumor resistance or Hh pathway modulators for regenerative medicine.