The mechanism for ligand activation of the Smoothened G protein–coupled receptor

The Smoothened (SMO) G protein–coupled receptor is a key part of the Hedgehog (Hh) signaling pathway, and it is an oncoprotein that is an important target for understanding cancers such as basal cell carcinoma. However, its mechanism of activation remains unknown. To this end, we investigate here the sequence of G protein and cholesterol (CHL) ligand binding to SMO on the pathway toward activation. Our results are consistent with the G protein–first activation pathway of SMO in the Hh signaling pathway. In this model, CHL first binds to the cysteine rich domain (CRD) of the inactive SMO, which remains inactive at this stage. The G protein can then spontaneously bind to this SMO, forming a precoupled complex that remains inactive while Patched (PTCH) is attached to the membrane. Upon binding of the Hh ligand to PTCH, the CHL levels in the membrane increase, enabling CHL to also bind to the transmembrane domain (TMD) of SMO. With CHL occupying both the CRD and the TMD sites, opening of the G protein becomes energetically favorable, promoting GDP release and initiating downstream G-protein signaling.