A Ciliary SMOOTHENED-GRK2-PKA Signaling Pathway Initiates Hedgehog Signal Transduction.

During Hedgehog (Hh) signal transduction in development, homeostasis, and cancer, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by directly binding the PKA catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh signal transduction in the vertebrate primary cilium. Hh pathway activation triggers rapid GRK2 relocalization from the base to the shaft of the cilium, leading to SMO phosphorylation and ultimately formation of SMO / PKA-C complexes in this compartment. In vitro reconstitution experiments reveal that GRK2 phosphorylation is sufficient to trigger direct binding of the SMO active conformation to PKA-C, without participation from additional proteins. Lastly, the SMO-GRK2-PKA communication pathway operates during Hh signaling in a range of cellular and in vivo models. Our work highlights GRK2 phosphorylation of ciliary SMO as a key initiating event for the intracellular steps of the Hh cascade, enabling a deeper understanding of how Hh signals are transduced intracellularly in tissues and organs to orchestrate proliferative and differentiative decisions. More broadly, our study hints at an expanded role for GRKs in enabling direct GPCR interactions with a diverse array of intracellular effectors.