Hedgehog regulates smoothened activity by inducing a conformational switch

Vol 450 | 8 November 2007 | doi:10.1038/nature06225 ARTICLES Hedgehog regulates smoothened activity by inducing a conformational switch Yun Zhao1*, Chao Tong1*{ & Jin Jiang1,2 Hedgehog (HH) morphogen is essential for metazoan development. The seven-transmembrane protein smoothened (SMO) transduces the HH signal across the plasma membrane, but how SMO is activated remains poorly understood. In Drosophila melanogaster, HH induces phosphorylation at multiple Ser/Thr residues in the SMO carboxy-terminal cytoplasmic tail, leading to its cell surface accumulation and activation. Here we provide evidence that phosphorylation activates SMO by inducing a conformational switch. This occurs by antagonizing multiple Arg clusters in the SMO cytoplasmic tail. The Arg clusters inhibit SMO by blocking its cell surface expression and keeping it in an inactive conformation that is maintained by intramolecular electrostatic interactions. HH-induced phosphorylation disrupts the interaction, and induces a conformational switch and dimerization of SMO cytoplasmic tails, which is essential for pathway activation. Increasing the number of mutations in the Arg clusters progressively activates SMO. Hence, by employing multiple Arg clusters as inhibitory elements counteracted by differential phosphorylation, SMO acts as a rheostat to translate graded HH signals into distinct responses. The HH morphogen controls many key development processes, with different thresholds specifying distinct outcomes1–4. In Drosophila wing discs, HH proteins secreted by posterior (P) compartment cells move into the anterior (A) compartment to form a local concentration gradient5,6. Low levels of HH suffice to induce the expression of decapentaplegic (dpp), whereas high levels are required to induce patched (ptc) and engrailed (en) (Supplementary Fig. 1)7–9. The reception system for HH consists of a twelve-transmembrane protein, PTC, as the HH receptor and a seven-transmembrane protein smoothened (SMO) as the signal transducer10–13. In Drosophila, HH binding to PTC abrogates its inhibition on SMO and induces extensive phosphorylation of the SMO cytoplasmic tail by protein kinase A (PKA) and casein kinase I (CKI), leading to SMO cell surface accumulation and activation14–17. How phosphorylation promotes SMO cell surface accumulation is not understood. In addition, phosphorylation may regulate SMO activity through mechanism(s) other than controlling its cell surface abundance. Regulation of SMO by multiple Arg clusters Our previous study indicates that phosphorylation may regulate SMO cell surface abundance by either preventing its endocytosis and/or promoting its recycling15. To investigate further how SMO cell surface expression is regulated, we generated a set of C-terminally truncated SMO variants and examined their subcellular localization using a cell-based assay (Fig. 1). Deletion up to amino acid 818 did not significantly change SMO subcellular distribution; however, further deletions resulted in progressively increased cell surface expression (Fig. 1a, c), implying that multiple negative regulatory elements exist between amino acids 661–818. SMODC710 exhibits consistently higher cell surface expression than SMODC730 (Fig. 1c), indicating that amino acids 710–730 may harbour a negative element(s). Ala-scan mutagenesis, which substituted multiple residues to Ala, identified the Arg residues in RRTQRRR as critical for preventing SMO cell surface accumulation (Fig. 1b, c; data not shown). Interestingly, multiple Arg clusters, arbitrarily named R1 to R4, are located between amino acids 661– 818, a region critical for blocking SMO cell surface accumulation (Fig. 1d). We therefore introduced into the full-length SMO Arg to Ala (RA) mutations in individual, or combinations of, Arg clusters. SMO variants with one Arg cluster mutated did not exhibit significant change in their cell surface expression; however, mutating two or more Arg clusters caused a gradual increase in SMO cell surface expression (Fig. 1d–f; data not shown), suggesting that multiple Arg clusters cooperate to restrict SMO cell surface accumulation. To determine whether the Arg clusters negatively regulate SMO activity, SMO variants with one or more mutated Arg clusters were expressed in wing discs using the MS1096 Gal4 driver. SMO variants with one mutated Arg cluster exhibited low levels of basal activity similar to that of wild-type SMO, as is evident from the ectopic expression of dpp but not ptc and en (Fig. 2a–c). However, SMO variants with two or more mutated Arg clusters exhibited a progressive increase in their constitutive signalling activities (Fig. 2d–i). Thus, SMO activity is inversely correlated with the number of functional Arg clusters. We also mutated several Arg clusters in the membrane-proximal region of the SMO cytoplasmic tail and observed no effect on SMO cell surface expression and activity (Supplementary Fig. 2). Hence, the Arg clusters between amino acids 661–818 are specifically involved in SMO autoinhibition. Phosphorylation counteracts the Arg motifs Increasing the number of phosphorylation-mimetic mutations in PKA/CKI sites resulted in a graded increase in SMO cell surface level and activity15, which phenocopies the effect of increasing the number of RA mutations, indicating that phosphorylation may activate SMO by antagonizing the Arg motifs. Consistently, an internal deletion that removes both the phosphorylation and Arg clusters (SMOD661– 818) results in high levels of SMO cell surface expression and activity (Figs 1a, c and 2j). It is intriguing that the Arg clusters are situated adjacent to the PKA/CKI phosphorylation clusters (Fig. 1d). In fact, R1, R2 and R4 1 Department of Developmental Biology, and 2Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. {Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA. *These authors contributed equally to this work. 252 ©2007 Nature Publishing Group ARTICLES NATURE | Vol 450 | 8 November 2007 are part of the PKA phosphorylation consensus site, R/KRXS. The juxtaposition of the Arg and phosphorylation clusters may allow precise control of SMO activity because phosphorylation at a c CFP SMON SMO∆C570 570 SMO∆C661 SMOWT 661 SMO∆C710 710 SMO∆C818 SMO∆C730 730 SMO∆C730 SMO∆C818 818 SMO∆C860 860 SMO∆C710 SMOWT 1035 SMO∆661–818 SMO∆C661 1035 b individual clusters may only neutralize the negative influence of adjacent Arg clusters. To test this, we constructed SMORA12D3 and found it behaved like SMORA124 (Fig. 1d, e; compare Fig. 2l with 2h), suggesting that phosphorylation at S3 (Fig. 1d) neutralizes the negative effect of R4. Because Arg carries positive charge whereas phosphorylation brings in negative charge, phosphorylation may antagonize the Arg clusters by neutralizing their positive charges. In support of this model, we found that R3 and R4 c (...truncated)