To investigate its function, we studied the sub-cellular localization of PTCHD1 and found that a PTCHD1-GFP fusion protein predominantly localizes to the cell membrane (Fig. 4b). We further hypothesized that PTCHD1 may function in the Hh-signaling pathway and have similar functional attributes as PTCH1 and PTCH2. We performed a Gli-dependent transcription assay in Hh-responsive 10T1/2 cells to test whether PTCHD1 could interfere with Hh signaling. In 10T1/2 cells, overexpression of PTCH1 or PTCH2 inhibits transcription from a Gli-luciferase reporter containing multiple copies of the Gli protein-binding site in the presence of Smoothened agonist purmorphamine (20) (Fig. 4c) or Gli2 (fig. S4). Similar to PTCH proteins, PTCHD1 also exerted a statistically significant inhibitory effect in these assays suggesting that PTCHD1 functions in the Hedgehog signalling pathway.
