Berger J, Patel H V, Woods J, Hayes N S, Parent S A, Clemas J, Leibowitz M D, Elbrecht A, Rachubinski R A, Capone J P, Moller D E
Department of Molecular Endocrinology, Merck Research Laboratories, Rahway, NJ 07065, USA.
Mol Cell Endocrinol. 2000 Apr 25;162(1-2):57-67. doi: 10.1016/s0303-7207(00)00211-2.
The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo
过氧化物酶体增殖物激活受体（PPARs）是核受体超家族的成员，作为配体激活的转录因子发挥作用。PPARγ在调节脂肪细胞分化和脂质代谢中起关键作用。最近，噻唑烷二酮（TZD）和某些非TZD抗糖尿病药物已被鉴定为PPARγ激动剂。为了进一步表征这个受体亚类，制备了一种缺少五个羧基末端氨基酸的突变型人PPARγ（hPPARγ2Delta500）。在用PPAR反应性报告构建体转染的COS-1细胞中，突变型受体不能被强效PPARγ激动剂激活。当与hPPARγ2或hPPARα共转染时，hPPARγ2Delta500以剂量反应方式消除野生型受体活性。hPPARγ2Delta500在高亲和力放射性配体结合方面也受损。此外，通过蛋白酶保护实验确定，其构象不受通常饱和浓度的PPARγ激动剂的影响。电泳迁移率变动分析表明，hPPARγ2Delta500和hPPARγ2都与人视黄醇X受体α（hRXRα）形成异二聚体复合物，并能以相似的亲和力结合过氧化物酶体增殖物反应元件（PPRE）。因此，hPPARγ2Delta500似乎通过与野生型受体竞争与RXR二聚化并结合PPRE来抑制PPAR活性。此外，突变型受体可能会耗尽PPAR调节的转录激活所需的因子。使用PPARγ特异性抗体通过免疫荧光测定，hPPARγ2和hPPARγ2Delta500都定位于瞬时转染的COS-1细胞的细胞核。因此，PPARγ的核定位与其激活状态无关。显性负突变体hPPARγ2Delta500可能在进一步研究中证明对体外和体内表征PPAR功能有用。
