A Structural Mechanism for Transduction of Hedgehog Signals Across the Membrane

The Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced across the membrane via an unusual mechanism: upon agonist-induced phosphorylation, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) binds directly to the protein kinase A (PKA) catalytic subunit (PKA-C) and physically blocks its enzymatic activity. How SMO binds PKA-C, and how phosphorylation triggers this process, remain obscure. Here we show, using computational structural methods combined with biochemical, biophysical, and functional approaches, that SMO engages PKA-C by mimicking strategies prevalent in canonical GPCR and PKA signaling complexes, despite little primary sequence or secondary structural homology. A PKA decoy substrate motif and amphipathic helix in the intrinsically disordered SMO intracellular domain bind the PKA-C active site cleft and hinge region, forming a SMO/PKA-C interface resembling the PKA regulatory subunit (PKA-R)/PKA-C holoenzyme. Meanwhile, the intracellular face of the SMO membrane-spanning domain engages a conserved PKA-C interaction hub in a manner strongly reminiscent of canonical GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key secondary structural elements within its cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural model for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.