Smoothened Signal Transduction Is Promoted by G Protein-Coupled Receptor Kinase 2

Abstract

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many “conventional” 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and β-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by β-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between β-arrestin 2 and Smoothened.

This work was supported by the SPORE program in the Pediatric Brain Tumor Foundation Institute at Duke University. This work was also supported in part by NIH grants N519576 and MH40159 to M.G.C. and HL706031 and HL16037 to R.J.L. M.G.C. is the NARSAD Lattner Foundation Distinguished Investigator. R.J.L. is an investigator for the Howard Hughes Medical Institute. A.R.M. was supported by the National Institute of General Medical Sciences (GM74349-01) and the Raychem/Rogers/Morris postdoctoral fellowship award. J.B.F. was supported by the National Institute of General Medical Sciences (GM069086).

We also thank Wei Chen, Trudy Oliver, and Xiao Dong Zhang for reagents; Martin Beaulieu and Ivan Medvedev for help with statistical analysis; Lynn Martinek for core fluorescence-activated cell sorter analysis support; and Amy Ramsey for helpful scientific discussions.