Characterizing the Structure and Function of the Cytoplasmic Domains of Ptch1

The Hedgehog (Hh)-pathway is a critical signalling cascade that determines cell fate and is required for embryonic and postnatal development. A central component of this molecular-genetic pathway is the receptor for the Hh-ligands, Patched-1 (Ptch1), a 12-pass, transmembrane glycoprotein that is essential for Hh-signalling. Despite its essential role in Hh-signalling, it is still unclear how Ptch1 mediates canonical Hh-signalling through Smo, though intracellular regions of Ptch1 have been suggested to be involved. We show that the deletion of the cytoplasmic â middle loopâ (ML) domain (a.a. 584-734) generates a Ptch1 mutant that, despite binding to ligand, constitutively suppresses Hh-signaling and increases the length of primary cilia. The constitutive activity due to deletion of this region is reversed, by further deletion of the cytoplasmic C-terminal domain (CTD) (a.a. 1162-1432). We show that the intracellular ML and CTD of Ptch1 are dispensable for canonical Hh-signalling.M.Sc