The Hedgehog (Hh) signaling pathway plays an essential and evolutionarily conserved role in patterning the embryonic body plan and in maintaining the proliferative status of adult cell populations. Inappropriate regulation of Hh signaling in vertebrates can lead to developmental defects and to multiple spontaneous and inherited cancers. One of the Hh pathway components implicated in the development of cancer is the transmembrane protein Smoothened (Smo). Smo is a primary determinant of Hh pathway activity, but the mechanism by which Smo mediates Hh signaling is not well understood. To identify the sequences necessary for Smo hyperphosphorylation, interaction with the downstream effector molecule Costal-2 (Cos2), and subcellular localization and to analyze the functional relevance of these events in Hh signaling, mutagenesis and biochemical characterization of Smo alleles will be conducted to map functional domains of Smo within its protein structure, and the cellular mediators of Smo phosphorylation will be identified and characterized through genetic and biochemical screening.