Opening the pore hinges on proline.

absence of Hh ligand, Ptc restrains Smo activity ; binding of Hh to Ptc releases Smo, which then transmits the intracellular signal. The regulation of Smo activity therefore occupies a pivotal position in the cellular response to Hh. Previous work had identified small molecules that inhibit or activate Hh signaling by interacting with Smo 6–8. These include the antagonist cyclopamine as well as further sets of structurally unrelated antagonists and an agonist. To identify the molecular target of purmor-phamine, Sinha and Chen took an elegant chemical genetics approach. Using a reporter assay for Hh-mediated transcriptional activation, they first demonstrated that Ptc is not required for purmorphamine activity. Cell lines derived from knockout mice lacking Ptc have constitutive Hh signaling. This is suppressed by the addition of cyclopamine; however, exposure to both cyclopamine and purmorphamine restored Hh signaling. Moreover, in normal cells, exposure to cyclo-pamine increased tenfold the concentration of purmorphamine necessary to elicit an Hh response. Conversely, compared to Sonic Hedgehog (Shh) treatment, purmorphamine significantly increased the dose of cyclopamine required to inhibit Hh signaling. The opposing activities of the two compounds strongly hinted that purmorphamine acted on Smo. Consistent with this, purmorphamine was unable to induce pathway activation in cells lacking Smo, but reintroduction of Smo restored purmorphamine sensitivity. Finally, the authors provided evidence that purmorphamine interacts directly with Smo by demonstrating that even in fixed cells, purmorphamine was able to compete with the binding of a fluorescent derivative of cyclopamine that had previously been shown to interact with Smo 6. Together, the studies of Sinha and Chen reveal that purmorphamine acts as an Hh signaling agonist by directly targeting Smo. Importantly, purmorphamine is structurally unrelated to the previously identified Smo agonist, thus establishing a new chemical class of Hh agonists. Despite the importance of Smo as the intra-cellular initiator of Hh signal transduction, uncertainty surrounds the mechanisms by which Smo activation is regulated and coupled to downstream events. The availability of purmorphamine offers new opportunities to tackle this question. The similarity of Smo to seven-transmembrane receptors raises the possibility that a conformational change is associated with activation; perhaps purmorphamine stabilizes Smo in its active state. But how is this switch in activity normally controlled? A clue comes from the structure of Ptc, which has similarity to the resistance-nod-ulation division family of permeases, bacterial drug/toxin efflux pumps 9. In addition, Ptc has a sterol-sensing domain (SSD), a cholesterol-binding motif …