Structural and Biophysical Characterization of Hedgehog Signaling

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DESCRIPTION (provided by applicant): Hedgehog (Hh) is a secreted signaling protein that plays a key role in the patterning of many tissues during animal development. Hh fits the classical definition of a morphogen by being expressed in specialized tissues and inducing cell growth and differentiation in neighboring tissues in a concentration dependent manner. Inappropriate Hh signaling leads to severe developmental abnormalities in embryos and is associated with many lethal cancers in adults. Because of its potent patterning activity, the distribution of both Hh and Hh responsiveness is tightly regulated. For example, Hh is dually lipidated and packaged into lipoprotein particles for secretion, and heparan sulfate proteoglycans are required for transport of Hh across multiple cell layers. At least 5 integral membrane proteins - Patched, Smoothened, Ihog, Dally-like protein, and Hedgehog-interacting protein - have been implicated in transducing or modulating Hh signals in Hh responsive cells. The overall goal of our studies is to understand the molecular interactions involved in Hh signaling and how these interactions are regulated during normal development. By understanding the nature of these interactions in normal circumstances, we also hope to understand how the pathway becomes activated in disease and how best to design inhibitors targeting the Hh pathway in such cases. Ihog is a type I integral membrane protein with multiple immunoglobulin and fibronectin type III (FNIII) repeats in its extracellular region. Hh has been shown to interact directly with one of the Ihog FNIII repeats in the presence of heparin, and we recently determined the crystal structure of a complex of Drosophila Hh and the Ihog FNIII domains. Heparin was unfortunately not visualized in this structure, and our first aim is to use biochemical, biophysical, and structural approaches to characterize the nature and role of the heparin involved in mediating interactions between Hh and Ihog. Curiously, the Hh binding site on vertebrate Ihog homologs occurs on a different Ihog FNIII domain than is observed for Drosophila Ihog, and our second aim is to characterize the interactions between vertebrate Hh and Ihog homologs and identify any dependence of this interaction on heparin or other co-factors. Our final aim is to express and purify the extracellular regions of each of the cell-surface components involved in Hh responsiveness and investigate the nature of any interactions among these components and Hh by biochemical, biophysical, and structural means.