PPAR-γ AF-2 domain functions as a component of a ubiquitin-dependent degradation signal

Abstract

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.