Turning Smo On

Abstract

Morphogens of the Hedgehog (Hh) family bind to the receptor Patched, and this alleviates inhibition of the signal-transducing membrane protein Smoothened. This activation of Smoothened involves increased cell surface abundance and phosphorylation of the cytoplasmic tail of Smoothened by protein kinase A and casein kinase I. By expressing Smoothened proteins with deletions or mutations in the cytoplasmic region, Zhao et al . found that multiple Arg clusters appeared responsible for limiting the cell surface accumulation of Smoothened expressed in S2 cells. Furthermore, the response was graded such that as more Arg clusters were mutated, the abundance of Smoothened at the surface progressively increased, and when these mutant Smoothened proteins were expressed in fly wing discs, the activity of Smoothened also was graded, with mutation of more Arg clusters leading to greater activity. The Arg clusters are adjacent to the phosphorylation sites, and mutations of the Arg clusters in combination with either mutation of the phosphorylation sites or introduction of phosphomimetic residues suggested that the negative charge created by phosphorylation activates Smoothened by neutralizing the positive charge of the Arg clusters. Fluorescence resonance energy transfer (FRET) experiments to detect intermolecular interactions suggested that Smoothened existed as a dimer constitutively (N-terminally tagged proteins produced a basal FRET signal) but that the cytoplasmic domains of the dimers did not interact (FRET produced by the cytoplasmic domains of C-terminally tagged proteins was low). Instead, the interaction between adjacent Smoothened cytoplasmic domains was increased by Hh. Proteins tagged to detect intramolecular interactions revealed that the C-terminal cytoplasmic domain was in close proximity to the third intracellular loop and that Hh decreased FRET between these. FRET analysis of the Smoothened mutants was consistent with phosphorylation adding a negative charge to mediate a change in the conformation of Smoothened. The affinity of the interaction between the Smoothened autoinhibitory domain (SAID) containing the Arg clusters and the phosphorylation sites and a minimal interacting fragment was progressively decreased by increasing phosphorylation or the introduction of increasing numbers of charge-neutralizing mutations. The authors propose that in the absence of Hh, Smoothened is a dimer and the cytoplasmic tail loops to form an inactive conformation mediated by the Arg clusters. Phosphorylation in response to Hh disrupts the cytoplasmic intramolecular interaction, allowing the cytoplasmic regions of the Smoothened dimer to interact. Whether increasing the number of sites phosphorylated triggers multiple active states or whether it increases the probability that the protein will adopt the active conformation remains unknown. Y. Zhao, C. Tong, J. Jiang, Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 450 , 252-258 (2007). [PubMed]