Structural mechanism for noncanonical GPCR signaling in the Hedgehog pathway

The Hedgehog (Hh) pathway is fundamental to embryogenesis, tissue homeostasis and cancer. Hh signals are transduced through an unusual mechanism; upon agonist-induced phosphorylation, the noncanonical G-protein-coupled receptor (GPCR) Smoothened (SMO) binds the protein kinase A (PKA) catalytic subunit (PKA-C) and physically blocks its enzymatic activity. Here, by combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary-structure homology. The intrinsically disordered SMO cytoplasmic domain binds the PKA-C active site, resembling the regulatory subunit within PKA holoenzymes, while the SMO transmembrane domain binds a conserved PKA-C interaction hub. Unlike prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize structural elements within its cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in Hh signaling and defines a principle for disordered GPCR domains to transmit signals intracellularly.