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Abstract
Patched-1 (Ptch1) is the primary receptor of the Hedgehog signalling pathway and mediates both canonical and non-canonical signalling. Previously our lab identified a group of proteins that interact with the C-terminus of murine Ptch1 that possessed SH3- and WW-binding domains, chief among them being C2-WW-HECT E3 ubiquitin ligases. Using a molecular-based approach, we showed that E3 ligases Smurf2 and Itch bind through sequences within the large intracellular middle loop and C-terminus of murine Ptch1 (mPtch1). We further demonstrated that mPtch1 is ubiquitinated in the presence of Smurf2 and Itch and this requires the middle loop and C-terminus. We also showed that mPtch1 can oligomerize and that the stability of mPtch1 is affected when the middle loop and C-terminus are deleted. Together, these data reveal a role for E3 ubiquitin ligases in mediating protein turnover of mPtch1 and show that the intracellular domains of mPtch1 interact with distinct protein factors.
