Abstract
The transcriptional coactivator PPARgamma coactivator-1alpha (PGC-1alpha) has been characterized as a broad regulator of cellular energy metabolism. Although PGC-1alpha functions through many transcription factors, the PGC-1alpha partners identified to date are unlikely to account for all of its biologic actions. The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) was identified in a yeast two-hybrid screen of a cardiac cDNA library as a novel PGC-1alpha-binding protein. ERRalpha was implicated previously in regulating the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD), which catalyzes the initial step in mitochondrial fatty acid oxidation. The cardiac perinatal expression pattern of ERRalpha paralleled that of PGC-1alpha and MCAD. Adenoviral-mediated ERRalpha overexpression in primary neonatal cardiac mycoytes induced endogenous MCAD expression. Furthermore, PGC-1alpha enhanced the transactivation of reporter plasmids containing an estrogen response element or the MCAD gene promoter by ERRalpha and the related isoform ERRgamma. In vitro binding experiments demonstrated that ERRalpha interacts with PGC-1alpha via its activation function-2 homology region. Mutagenesis studies revealed that the LXXLL motif at amino acid position 142-146 of PGC-1alpha (L2), necessary for PGC-1alpha interactions with other nuclear receptors, is not required for the PGC-1alpha.ERRalpha interaction. Rather, ERRalpha binds PGC-1alpha primarily through a Leu-rich motif at amino acids 209-213 (Leu-3) and utilizes additional LXXLL-containing domains as accessory binding sites. Thus, the PGC-1alpha.ERRalpha interaction is distinct from that of other nuclear receptor PGC-1alpha partners, including PPARalpha, hepatocyte nuclear factor-4alpha, and estrogen receptor alpha. These results identify ERRalpha and ERRgamma as novel PGC-1alpha interacting proteins, implicate ERR isoforms in the regulation of mitochondrial energy metabolism, and suggest a potential mechanism whereby PGC-1alpha selectively binds transcription factor partners.
Highlights
The transcriptional coactivator PPAR␥ coactivator-1␣ (PGC-1␣) has been characterized as a broad regulator of cellular energy metabolism
This region of the PGC-1␣ protein contains all of the domains known to mediate interactions between PGC-1␣ and transcription factors partners identified to date [21]
The PGC-1␣1⁄7ERR␣ Interaction Interface Is Distinct from That of Other Nuclear Receptor PGC-1␣ Partners—The results shown above suggest that the PGC-1␣1⁄7ERR␣ interaction involves the L3 site, a novel PGC-1␣/nuclear receptor interface
Summary
CV1, 293, and HepG2 cells were maintained at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium/10% fetal calf serum. HepG2 cells were plated the day before transfection in Earle’s minimum essential medium without phenol red/ 10% stripped fetal calf serum. Reporter plasmids (4 g/ml) were cotransfected with Rous sarcoma virus--galactosidase (0.5 g/ml), expressing the -galactosidase gene driven by the Rous sarcoma virus promoter, to control for transfection efficiency. After 24 h cells were infected with adenovirus expressing GFP (Ad-GFP) or ERR␣ (Ad-ERR␣) driven by a cytomegalovirus promoter. The latter construct expresses GFP from an independent promoter. Recombination and propagation of adenovirus expressing ERR␣ was performed as described [17]
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