PPAR‐γ AF‐2 Domain Functions as a Component of a Ubiquitin‐dependent Degradation Signal

The nuclear hormone receptor peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) functions as the “master switch” in adipocyte development and is important in regulating glucose metabolism. PPAR‐γ is rapidly degraded in adipocytes by the ubiquitin proteasome pathway under basal and ligand‐activated conditions. Proteasome inhibition increases PPAR‐γ activity, indicating disposal of PPAR‐γ by the ubiquitin proteasome system regulates PPAR‐γ activity. However, the signals and factors required for recognition of PPAR‐γ by the ubiquitin proteasome pathway are unknown. To begin understanding how the ubiquitin‐proteasome pathway interacts with PPAR‐γ, we designed a series of constructs containing each PPAR‐γ domain expressed as a fusion protein with the GAL4 DNA‐binding domain. The ability of each PPAR‐γ domain to alter the stability of the GAL4 DNA‐binding domain and to undergo ubiquitylation was assessed via western blot analysis. In addition, luciferase reporter assays were used to assay PPAR‐γ transcriptional activity. Using this approach, we determined that the AF‐1 and ligand‐binding domains (LBDs) of PPAR‐γ are targeted to the proteasome for degradation. However, only the LBD is conjugated to ubiquitin. The AF‐2 helix of the LBD is required for maximum ubiquitylation, but is not essential for ligand‐dependent ubiquitin conjugation. Finally, luciferase reporter assays show a fully functional ubiquitin system is required for PPAR‐γ activation. These results indicate that the ubiquitin‐proteasome pathway is an integral determinant of PPAR‐γ activity, targeting PPAR‐γ for proteasomal degradation via ubiquitin independent and ubiquitin dependent mechanisms.