The Localization Of Smoothened In Primary Cilia Is Regulated By Its Transmenbrane Domain

Sonic Hedgehog (Shh) is an important morphogen that governs cell growth and patterning in numerous developmental processes in both vertebrates and invertebrates. Shh signalling pathway has the critical biological roles in many aspects of tissue patterning such as limb, neural system, bone, heart, lung, genital system and skin. Mutations in various components of this pathway frequently occur in tumors and abnormal activation of this pathway is associated with lots of cancers.In the absence of Hh ligand, Ptch is located at the ciliary membrane, where it inhibits the formation of active Smo from ciliary membrane. Without active Smo the Gli transcription factors are stepwise labeled by phosphorylation and ubiquitination events for proteosomal cleavage into a truncated repressor form (GliR). GliR travels into the nucleus and inhibits gene transcription of Hh-target-genes. In the presence of the Hh ligand, it binds to Ptch, active Smo is formed at the ciliary membrane and regulate Gli-Sufu complex to the tip of cilia. Now the Gli transcription factors can be processed to their active forms (GliA) that are transported to the nucleus where they finally activate gene transcription of the Hh-target-genes.Inappropriate activation of the Shh response pathway is associated with several types of human tumor, loss-of-function mutations of Ptch or activating mutations of Smo are found in around40%of sporadic BCC and25%of primitive neuroectodermal. Recently scientists have found a class of steroidal alkaloid include of cyclopamine from natural plant that can inhibit Smo activity. Up to now cyclopamime has been into the stage of clinical trial as the experimental drug to treat basal cell carcinoma and medulloblastoma.Smo is a seven transmembrane protein having three domains:N-extracellular domain, seven-transmenbrane domain and C-intracellular domain. Nowadays, many researches hold the opinion that the C-terminal domain is the main domain for its activity. They think the phosphorylation of Smo is mainly in C-terminal, Smo adopt an active conformation called dimer through its C-terminal to enter into the primary cilia then activate the pathway. They also think that Smo activity is controlled by its C-terminal domain, the longer the C-terminal domain the more activity Smo may get. But until now,the functions of Smo seven-transmenbrane domain and how does this domain regulate the ciliary localization of Smo remain a deep mystery.To investigate these questions, we firstly studied the effect of Smo seven-transmembrane domain on the protein stability. We constructed SDTm plasmids that contains different Smo transmembrane domain and marked their C terminal with GFP or Flag tag. We found SDTm1-6which only has Smo7th transmenbrane domain was less stable than full length Smo, but other SDTm were as stable as full length Smo. We further found that because SDTm1-6is much shorter then Smo full length, it was degraded by proteasome quickly.Since all SDTm except SDTml-6are as stable as Smo full length, we wonder whether the seven-transmenbrane domin plays a role in the signal transduction. By Gli-Luc reporter assay, we found that all SDTm could raise the Gli-Luc values, indicating that SDTm can coordinated with endogenous Smo on activating the pathway. Further more when we transfected Smofl/fl cells with Ad-Cre-GFP to knock down endogenous Smo, we found that SDTm can still raise the Gli-Luc values. These results indicate that the seven-transmenbrane domain of Smo has no influence in Shh signal transduction.At last, we studied the effect of seven-transmembrane domain on ciliary localization of Smo. The result showed that when lack of the Smo lth-2th transmenbrane domain (SDTml-2), Smo can still localize in the primary cilia; but when we cut6th-7th transmenbrane domain (SDTm6-7), Smo could not go into the primary cilia. All of these data indicate that Smo transmenbrane domain plays an important role in regulating the ciliary localization of Smo.From all above mentioned, we hold the opinion that Smo seven-transmenbrane domain did not act on Smo protein stability and Shh signaling transduction, but was critical to the localization of Smo in primary cilia. In this study, we partly explained the molecular mechanism of Smo seven-transmembrane domain on regulating ciliary localization of Smo. Understanding the molecular mechanism of Shh signaling transduction can help us to find the new targets to regulate development and cancer.