Regulation of Smoothened ubiquitination and cell surface expression by a Cul4-DDB1-Gβ E3 ubiquitin ligase complex

Abstract

Hedgehog (Hh) transduces signal by promoting cell surface accumulation and activation of the G protein coupled receptor (GPCR)-family protein Smoothened (Smo) in Drosophila, but the molecular mechanism underlying the regulation of Smo trafficking has remained poorly understood. Here we identify a Cul4-DDB1 E3 ubiquitin ligase complex as essential for Smo ubiquitination and cell surface clearance. We find that the C-terminal intracellular domain of Smo recruits Cul4-DDB1 through the β subunit of trimeric G protein (Gβ), and that Cul4-DDB1-Gβ promotes the ubiquitination of both Smo and its binding partner G-protein-coupled-receptor-kinase 2 (Gprk2) and induces the internalization and degradation of Smo. Hh dissociates Cul4-DDB1 from Smo by recruiting the catalytic subunit of protein kinase A (PKA) to phosphorylate DDB1, which disrupts its interaction with Gβ. Inactivation of the Cul4-DDB1 complex resulted in elevated Smo cell surface expression whereas excessive Cul4-DDB1 blocked Smo accumulation and attenuated Hh pathway activation. Taken together, our study identifies an E3 ubiquitin ligase complex targeting Smo for ubiquitination and provides new insight into how Hh signaling regulates Smo trafficking and cell surface expression.