The GPCR-like protein Smoothened involved in the Hedgehog pathway : Signalling properties of variants resulting from somatic mutations

Abstract

The Hedgehog (Hh) signalling pathway is essential for numerous processes during embryonic development including development of the skin and pancreas. Dysregulation of Hh-signalling during development might be lethal and may lead to cancer in adult cells. This pathway is important in several types of cancers including cancers in the pancreas, brain and skin. The best known examples of excessive Hh-signalling causing cancer are the frequent mutations and dysregulation observed in basal cell carcinoma (BCC), a type of skin cancer. The Hh-pathway is considered as a very promising target for new anti-cancer treatments and increased knowledge of this pathway may lead to new strategies of cancer treatment.

Hedgehog (Hh), Patched (Ptch), Smoothened (Smo), Suppressor of fused (Sufu), fused (Fu) and Gli are signalling proteins in the Hh-pathway. Mutations in genes encoding these members of the Hh signalling pathway may lead to cancer. Smoothened is a seven transmembrane protein which has been studied in this current project and shows several characteristics of being a G-protein-coupled receptor (GPCR). Fourteen somatic mutations in Smo have been identified. Five of these mutations have been analysed herein and one of these mutations was generated as part of the thesis work (K575M-Smo).

First, PCR-based mutagenesis was performed to generate a mutated version of Smo, K575M-Smo. Subsequently the PCR-product containing K575M-Smo was inserted into the pEF.6 vector.

Secondly, K575M-Smo, four other mutants (R484W-, L514F-, S533N- and W535L-Smo) and Smo wt were sub-cloned from the pEF.6 vector into the mammalian expression vector p3xFLAG-CMV-10.

NIH/3T3 mouse fibroblast cells were stably transfected with the following constructs: p3xFLAG-CMV-10 vector (empty vector control), p3xFLAG-CMV-10 R484W-, L514F-, S533N-, W535L-, K575M-Smo and Smo wt, to analyze the signalling properties of the different mutated versions of Smo. From each transfection, six independent monoclonal cell lines potentially harbouring the transfected plasmid were isolated, and different experiments were performed, including real-time reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and reporter gene assay.

Taken together, the K575M-Smo encoding plasmid was successfully generated in the laboratory. Initial experiments showed a trend of constitutive activity of W535L-Smo. Based on the variable data from real-time RT-PCR experiments, it is not possible to make conclusions about the signalling properties of the various Smo mutations, and further studies are needed.