A PPAR gamma mutant serves as a dominant negative inhibitor of PPAR signaling and is localized in the nucleus

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription fa ctors. PPAR gamma plays a critical role in regulating adipocyte differentia tion and lipid metabolism. Recently, thiazolidinedione (TZD) and select non -TZD antidiabetic agents have been identified as PPAR gamma agonists. To fu rther characterize this receptor subclass, a mutant hPPAR gamma lacking fiv e carboxyl-terminal amino acids was produced (hPPAR gamma 2 Delta 500). In COS-I cells transfected with PPAR-responsive reporter constructs, the mutan t receptor could not be activated by a potent PPAR gamma agonist. When cotr ansfected with hPPAR gamma 2 or hPPAR alpha, hPPAR gamma 2 Delta 500 abroga ted wild-type receptor activity in a dose-responsive manner. hPPAR gamma 2 Delta 500 was also impaired with respect to binding of a high-affinity radi oligand. In addition, its conformation was unaffected by normally saturatin g concentrations of PPAR gamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPAR gamma 2 Delta 500 and hPPAR gamma 2 both formed heterodimeric complexes wi th human retinoid x receptor alpha (hRXR alpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, h PPAR gamma 2 Delta 500 appears to repress PPAR activity by competing with w ild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcr iptional activation. Both hPPAR gamma 2 and hPPAR gamma 2 Delta 500 localiz ed to the nucleus of transiently transfected COS-1 cells as determined by i mmunofluorescence using a PPAR gamma-specific antibody. Thus, nuclear local ization of PPAR gamma occurs independently of its activation state. The dom inant negative mutant, hPPAR gamma 2 Delta 500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.