Sonic Hedgehog Activates Phospholipase A2 to Enhance Smoothened Ciliary Translocation

The G protein-coupled receptor Smoothened (Smo) is the signal transducer of the Sonic Hedgehog (Shh) pathway. Smo signals through G proteindependent and -independent routes, with G protein-independent canonical signaling to Gli effectors requiring Smo accumulation in the primary cilium. The mechanisms controlling Smo activation and trafficking are not yet clear but likely entail small-molecule binding to pockets in its extracellular cysteine-rich domain (CRD) and/or transmembrane bundle. Here, we demonstrate that the cytosolic phospholipase cPLA2 alpha is activated through Gb gamma downstream of Smo to release arachidonic acid. Arachidonic acid binds Smo and synergizes with CRD-binding agonists, promoting Smo ciliary trafficking and high-level signaling. Chemical or genetic cPLA2 alpha inhibition dampens Smo signaling to Gli, revealing an unexpected contribution of G proteindependent signaling to canonical pathway activity. Arachidonic acid displaces the Smo transmembrane domain inhibitor cyclopamine to rescue CRD agonist-induced signaling, suggesting that arachi-donic acid may target the transmembrane bundle to allosterically enhance signaling by CRD agonist-bound Smo.