Peroxisome Proliferator-activated Receptor Coactivator-1α (PGC-1α) Coactivates the Cardiac-enriched Nuclear Receptors Estrogen-related Receptor-α and -γ

The transcriptional coactivator PPARγ coactivator-1α (PGC-1α) has been characterized as a broad regulator of cellular energy metabolism. Although PGC-1α functions through many transcription factors, the PGC-1α partners identified to date are unlikely to account for all of its biologic actions. The orphan nuclear receptor estrogen-related receptor α (ERRα) was identified in a yeast two-hybrid screen of a cardiac cDNA library as a novel PGC-1α-binding protein. ERRα 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 ERRα paralleled that of PGC-1α and MCAD. Adenoviral-mediated ERRα overexpression in primary neonatal cardiac mycoytes induced endogenous MCAD expression. Furthermore, PGC-1α enhanced the transactivation of reporter plasmids containing an estrogen response element or the MCAD gene promoter by ERRα and the related isoform ERRγ. In vitro binding experiments demonstrated that ERRα interacts with PGC-1α via its activation function-2 homology region. Mutagenesis studies revealed that the LXXLL motif at amino acid position 142–146 of PGC-1α (L2), necessary for PGC-1α interactions with other nuclear receptors, is not required for the PGC-1α·ERRα interaction. Rather, ERRα binds PGC-1α 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-1α·ERRα interaction is distinct from that of other nuclear receptor PGC-1α partners, including PPARα, hepatocyte nuclear factor-4α, and estrogen receptor α. These results identify ERRα and ERRγ as novel PGC-1α interacting proteins, implicate ERR isoforms in the regulation of mitochondrial energy metabolism, and suggest a potential mechanism whereby PGC-1α selectively binds transcription factor partners. The transcriptional coactivator PPARγ coactivator-1α (PGC-1α) has been characterized as a broad regulator of cellular energy metabolism. Although PGC-1α functions through many transcription factors, the PGC-1α partners identified to date are unlikely to account for all of its biologic actions. The orphan nuclear receptor estrogen-related receptor α (ERRα) was identified in a yeast two-hybrid screen of a cardiac cDNA library as a novel PGC-1α-binding protein. ERRα 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 ERRα paralleled that of PGC-1α and MCAD. Adenoviral-mediated ERRα overexpression in primary neonatal cardiac mycoytes induced endogenous MCAD expression. Furthermore, PGC-1α enhanced the transactivation of reporter plasmids containing an estrogen response element or the MCAD gene promoter by ERRα and the related isoform ERRγ. In vitro binding experiments demonstrated that ERRα interacts with PGC-1α via its activation function-2 homology region. Mutagenesis studies revealed that the LXXLL motif at amino acid position 142–146 of PGC-1α (L2), necessary for PGC-1α interactions with other nuclear receptors, is not required for the PGC-1α·ERRα interaction. Rather, ERRα binds PGC-1α 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-1α·ERRα interaction is distinct from that of other nuclear receptor PGC-1α partners, including PPARα, hepatocyte nuclear factor-4α, and estrogen receptor α. These results identify ERRα and ERRγ as novel PGC-1α interacting proteins, implicate ERR isoforms in the regulation of mitochondrial energy metabolism, and suggest a potential mechanism whereby PGC-1α selectively binds transcription factor partners. Cellular energy production is tightly linked to metabolic demand, which is, in turn, dictated by diverse developmental, physiologic, and environmental conditions. The capacity for cellular ATP production is controlled, in part, by the expression levels of nuclear genes involved in mitochondrial oxidative metabolism. Thus, tight regulation of cellular energy metabolism necessitates transduction of diverse signals related to cellular energy demands to the nucleus. Although numerous factors involved in the transcriptional regulation of metabolic gene expression have been identified, the precise pathways involved in the physiologic control of cellular energy metabolism have not been delineated. The recent discovery of PPARγ coactivator-1α (PGC-1α), 1The abbreviations used are: PGC-1α, PPARγ coactivator-1α; BAT, brown adipose tissue; PPAR, peroxisome proliferator-activated receptor; FAO, fatty acid oxidation; MCAD, medium-chain acyl-CoA dehydrogenase; ERR, estrogen-related receptor; GFP, green fluorescent protein; COUP-TF, chicken ovalbumin upstream promoter transcription factor; GST, glutathioneS-transferase; aa, amino acid; LBD, ligand binding domain; DBD, DNA binding domain; NRRE, nuclear receptor response element; MAPK, mitogen-activated protein kinase; NR, nuclear receptor; HNF-4α, hepatocyte nuclear factor-4α; ERα, estrogen receptor α. 1The abbreviations used are: PGC-1α, PPARγ coactivator-1α; BAT, brown adipose tissue; PPAR, peroxisome proliferator-activated receptor; FAO, fatty acid oxidation; MCAD, medium-chain acyl-CoA dehydrogenase; ERR, estrogen-related receptor; GFP, green fluorescent protein; COUP-TF, chicken ovalbumin upstream promoter transcription factor; GST, glutathioneS-transferase; aa, amino acid; LBD, ligand binding domain; DBD, DNA binding domain; NRRE, nuclear receptor response element; MAPK, mitogen-activated protein kinase; NR, nuclear receptor; HNF-4α, hepatocyte nuclear factor-4α; ERα, estrogen receptor α. PGC-1β, and the PGC-1-related protein, a family of inducible transcriptional coactivators responsive to selective physiological stimuli, have provided new insights into the link between extracellular events and the regulation of genes involved in energy metabolism. PGC-1α, the first member of this novel coactivator family to be identified, was initially characterized as a key regulator of thermogenesis in brown adipose tissue (BAT) and skeletal muscle via its coactivation of the adipose-enriched nuclear receptor, PPARγ (1Puigserver P., Wu, Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Google Scholar, 2Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Google Scholar). Subsequent studies have revealed a broader role for PGC-1α in a variety of cellular energy metabolic processes including mitochondrial biogenesis, mitochondrial fatty acid oxidation (FAO), and gluconeogenesis (2Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Google Scholar, 3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar, 4Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D. Kelly D.P. J. Clin. Invest. 2000; 106: 847-856Google Scholar, 5Michael L.F., Wu, Z. Cheatham R.B. Puigserver P. Adelmant G. Lehman J.J. Kelly D.P. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3820-3825Google Scholar, 6Yoon J.C. Puigserver P. Chen G. Donovan J., Wu, Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Google Scholar). The function of PGC-1β and PGC-1-related protein remain to be defined.PGC-1α is unique from the p160 and p300/cAMP response element-binding protein-binding protein classes of transcriptional coactivators in its tissue-restricted expression pattern, its developmental regulation, and its inducibility by specific physiological stimuli. PGC-1α is enriched in tissues reliant on oxidative metabolism for ATP generation (heart, skeletal muscle) or heat (BAT) but is also expressed in liver, brain, and kidney (1Puigserver P., Wu, Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Google Scholar). Immediately after birth, PGC-1α expression increases in heart coincident with a shift from reliance on glycolysis to mitochondrial FAO as the chief energy source in the adult myocardium (4Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D. Kelly D.P. J. Clin. Invest. 2000; 106: 847-856Google Scholar). PGC-1α expression is induced in adult skeletal muscle, BAT, and heart in response to stimuli that increase energy demands. For example, cold exposure leads to a rapid induction of PGC-1α gene expression in BAT (1Puigserver P., Wu, Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Google Scholar, 7Boss O. Bachman E. Vidal-Puig A. Zhang C.-Y. Peroni O. Lowell B.B. Biochem. Biophys. Res. Commun. 1999; 261: 870-876Google Scholar). In addition, fasting and short-term exercise induces PGC-1α gene expression in heart and skeletal muscle, respectively (4Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D. Kelly D.P. J. Clin. Invest. 2000; 106: 847-856Google Scholar,8Goto M. Terada S. Kato M. Katoh M. Yokozeki T. Tabata I. Shimokawa T. Biochem. Biophys. Res. Commun. 2000; 274: 350-354Google Scholar, 9Baar K. Wende A.R. Jones T.E. Marison M. Nolte L.A. Chen M. Kelly D.P. Holloszy J.O. FASEB J. 2002; (in press)Google Scholar). Recent studies have shown that PGC-1α protein is phosphorylated in response to cytokine stimulation of the p38 mitogen-activated protein kinase pathway resulting in stabilization of the protein (10Puigserver P. Rhee J. Lin J., Wu, Z. Yoon J.C. Zhang C.-Y. Kraquss S. Mootha V.K. Lowell B.B. Spiegelman B.M. Mol. Cell. 2001; 8: 971-982Google Scholar). Furthermore, we have shown that activation of the p38 pathway enhances ligand-dependent PGC-1α coactivation of PPARα (11Barger P.M. Browning A.C. Garner A.N. Kelly D.P. J. Biol. Chem. 2001; 276: 44495-44501Google Scholar). Finally, Knutti et al. (12Knutti D. Kressler D. Kralli A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9713-9718Google Scholar) demonstrated p38-mediated activation of PGC-1α via release of a repressor.The results of recent gain-of-function studies have demonstrated that PGC-1α serves as a global regulator of mitochondrial metabolic capacity. Spiegelman and co-workers (2Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Google Scholar) have shown that overexpression of PGC-1α in myogenic cells activates the mitochondrial biogenic program leading to an increase in mitochondrial number and respiration rates. Our laboratory has shown that forced expression of PGC-1α in primary cardiac myocytes and in hearts of transgenic mice leads to the transcriptional activation of genes encoding mitchondrial FAO enzymes, such as medium-chain acyl-CoA dehydrogenase (MCAD), and triggers a robust increase in mitochondrial cellular volume density (4Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D. Kelly D.P. J. Clin. Invest. 2000; 106: 847-856Google Scholar). Collectively, these recent studies suggest that PGC-1α serves to transduce stimuli linked to physiologic demands to the transcriptional control of mitochondrial functional capacity.The regulatory effects of PGC-1α are thought to be mediated primarily by its ability to interact with and coactivate numerous nuclear receptors, as well as non-nuclear receptor transcription factors, based on in vitro and cell culture studies (1Puigserver P., Wu, Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Google Scholar, 2Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Google Scholar, 3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar, 6Yoon J.C. Puigserver P. Chen G. Donovan J., Wu, Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Google Scholar, 12Knutti D. Kressler D. Kralli A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9713-9718Google Scholar, 13Tcherepanova I. Puigserver P. Norris J.D. Speigelman B.M. McDonnell D.P. J. Biol. Chem. 2000; 275: 16302-16308Google Scholar, 14Delerive P., Wu, Y. Burris T.P. Chin W.W. Suen C.S. J. Biol. Chem. 2002; 277: 3913-3917Google Scholar). The effects of PGC-1α on mitochondrial FAO enzyme gene expression occur, at least in part, through its activation of the nuclear receptor PPARα (3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar, 4Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D. Kelly D.P. J. Clin. Invest. 2000; 106: 847-856Google Scholar). The mitochondrial biogenesis response involves the transcription factors nuclear respiratory factor-1 and nuclear respiratory factor-2 (2Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Google Scholar). However, not all of the downstream effects of PGC-1α on cellular energy metabolism have been ascribed to particular PGC-1α transcription factor partners. The role of PGC-1α as a “master” regulator of cellular energy metabolism is likely mediated by multiple transcription factors, some of which could be novel. In addition, the mechanisms involved in partner selection by PGC-1α in the context of coexpressed transcription factors are also unknown.In an attempt to identify PGC-1α interacting proteins relevant to the postnatal heart, we performed a two-hybrid screen of an adult human heart cDNA library in yeast using PGC-1α as “bait.” We focused on the adult mammalian heart because of its extraordinary capacity to match mitochondrial energy production with high demands. The orphan nuclear receptor, estrogen-related receptor α (ERRα; NR3B1), was identified as a novel PGC-1α interacting protein. PGC-1α enhanced the transcriptional activities of ERRα and the related isoform ERRγ. The PGC-1α·ERRα complex was shown to directly activate the MCAD promoter, a gene implicated previously as an ERRα target. Functional assays and in vitro binding studies demonstrated that ERRα binds PGC-1α via a novel set of leucine-rich domains compared with other characterized nuclear receptor PGC-1α partners. These results identify a novel PGC-1α target in heart. In addition, our results suggest that the utilization of distinct binding interfaces within PGC-1α provides one mechanism whereby this versatile coactivator may differentially activate multiple downstream effectors in diverse cellular and physiologic contexts.EXPERIMENTAL PROCEDURESMammalian Cell Culture and Transient TransfectionsCV1, 293, and HepG2 cells were maintained at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium/10% fetal calf serum. For experiments requiring estradiol, HepG2 cells were plated the day before transfection in Earle's minimum essential medium without phenol red/10% stripped fetal calf serum. Transient transfections were performed by the calcium phosphate coprecipitation method as described (15Disch D.L. Rader T.A. Cresci S. Leone T.C. Barger P.M. Vega R. Wood P.A. Kelly D.P. Mol. Cell. Biol. 1996; 16: 4043-4051Google Scholar). Reporter plasmids (4 μg/ml) were cotransfected with Rous sarcoma virus-β-galactosidase (0.5 μg/ml), expressing the β-galactosidase gene driven by the Rous sarcoma virus promoter, to control for transfection efficiency. For cotransfection experiments, mammalian expression vectors (see below) for nuclear receptors, PGC-1α constructs, or the corresponding empty vectors were used. Ligands were added immediately following transfection protocol at the concentrations indicated in the figure legends, and cells were collected and assayed 48 h later. Luciferase and β-galactosidase activities were measured as described (16Brandt J. Djouadi F. Kelly D.P. J. Biol. Chem. 1998; 273: 23786-23792Google Scholar).Ventricular cardiac myocytes were prepared from 1-day-old Harlan Sprague-Dawley rats as described (15Disch D.L. Rader T.A. Cresci S. Leone T.C. Barger P.M. Vega R. Wood P.A. Kelly D.P. Mol. Cell. Biol. 1996; 16: 4043-4051Google Scholar). After 24 h cells were infected with adenovirus expressing GFP (Ad-GFP) or ERRα (Ad-ERRα) driven by a cytomegalovirus promoter. The latter construct also expresses GFP from an independent promoter. Infection rate of 90–95% was achieved by 18 h as assessed by quantitation of GFP-expressing cells using fluorescence microscopy. Whole cell protein extracts were prepared from cells 72 h post-infection. The adenoviral construct, Ad-ERRα, was constructed by subcloning aSalI/NotI fragment from the pBK-ERRα (generously provided by C. Teng) construct containing the full-length human ERRα cDNA encoding amino acids 1–422 into the pAd-Track-cytomegalovirus vector. Recombination and propagation of adenovirus expressing ERRα was performed as described (17He T.C. Zhou S. da Costa L.T., Yu, J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Google Scholar).Plasmid ConstructsReporter PlasmidsThe MCAD promoter-luciferase plasmids have been described (15Disch D.L. Rader T.A. Cresci S. Leone T.C. Barger P.M. Vega R. Wood P.A. Kelly D.P. Mol. Cell. Biol. 1996; 16: 4043-4051Google Scholar, 18Gulick T. Cresci S. Caira T. Moore D.D. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11012-11016Google Scholar). The Vit2P36.Luc, containing two copies of the vitellogenin estrogen responsive element upstream of the prolactin minimal promoter, was generously provided by S. Adler (Southern Illinois University). The (UAS)3.TK.Luc was a gift from D. Moore (Baylor College of Medicine). The (APOCIII)2.TK.Luc was constructed by ligation of annealed oligonucleotides (sense strand 5′-GATCCTCATCTCCACTGGTCAGCAGGTGACCTTTGCCCAGCGCCCTGGGA-3′) into the BamHI/BglII site upstream of the thymidine kinase promoter of the pGL2-TK.Luc reporter plasmid. The sequence is based on the HNF-4 response element contained in the human apolipoprotein CIII gene promoter (19Ladias J.A.A. Hadzopoulou-Cladaras M. Kardassis D. Cardot P. Cheng J. Zannis V. Cladaras C. J. Biol. Chem. 1992; 267: 15849-15860Google Scholar).Mammalian Expression PlasmidsThe mammalian expression vector pcDNA3.1-Myc/His.PGC-1α has been described elsewhere (3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar). Site-directed mutations in PGC-1α were introduced by a PCR-based strategy (Quickchange Mutagenesis kit; Stratagene) using the Myc/His.PGC-1α as template for single-site mutants. The double- and triple-site mutations were made using the single- and double-mutant templates, respectively. The PGC-1α deletion series, PGC338, PGC285, and PGC120, have been described (3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar). Additional FLAG-tagged PGC-1α deletion constructs were generated by PCR introducing an in-frame BglII at the start codon and a stop codon at 273, 213, 208, or 191. PCR fragments were then subcloned (BglII/XhoI) into cytomegalovirus promoter-Tag1 (Stratagene) to fuse a cassette encoding a FLAG epitope to 5′ end of the PGC-1α sequence. The PGC-1α constructs were subsequently cloned into the NotI site of the pcDNA3.1 mammalian vector for cotransfection studies. The pcDNA3.1-FLAG-ERRα full-length and deletion constructs were generated by the same procedure described above for PGC-1α using PCR to introduce a stop codon at 403, 359, or 209. The Gal4-ERRα and Gal4-ERRα403 were generated by subcloning theBamHI fragment from the corresponding pcDNA3.1 construct into the pCMX-Gal4 plasmid (a kind gift from D. Moore). The Gal4-PPARα has been described (3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar). The pSG5-hemagglutinin-ERRγ, the pBK-Rous sarcoma virus-ERα, and the pMT-HNF-4 expression vectors were kind gifts from M. Stallcup (University of Southern California), S. Adler, and J. Ladias (Harvard Medical School), respectively.Bacterial Expression PlasmidsConstruction of the pGex4T- 3-PGC338 has been described elsewhere (3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar). The pGex4T-3-ERRα was generated by subcloning the BamHI fragment from the pcDNA3.1-ERRα into the pGex4T-3 vector (Amersham Biosciences).Northern Blot AnalysisTotal cellular RNA isolation and blotting was performed as described (16Brandt J. Djouadi F. Kelly D.P. J. Biol. Chem. 1998; 273: 23786-23792Google Scholar). Blots were hybridized with radiolabeled probes derived from the following cDNA mouse clones: MCAD, ERRγ, PPARα, and PGC-1α. In addition, human ERRα, rat M-CPT I, and universal actin probes were used.ImmunoblottingProtein extracts were resolved by SDS-PAGE (7.5%). Transfer and detection were performed as described (20Huss J.M. Levy F.H. Kelly D.P. J. Biol. Chem. 2001; 276: 27605-27612Google Scholar). Immunodetection of ERRα and COUP-TF were performed using polyclonal anti-ERRα or anti-COUP-TF antibodies generously provided by V. Giguere (McGill University) and M.-J. Tsai (Baylor College of Medicine), respectively. MCAD was detected using the anti-MCAD antibody described previously.GST Pull Down AssaysIn vitro protein-protein interaction assays have been described previously (3Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar). 35S-Labeled proteins were synthesized in the TNT T7 quick-coupled in vitrotranscription/translation system (Promega). In pull down reactions, 50 μl of a 50% slurry of GST fusion protein bound to glutathione-Sepharose was incubated with 10 μl of35S-labeled protein in 500 μl of binding buffer (20 mm Tris, 7.5, 100 mm KCl, 0.1 mmEDTA, 0.05% Nonidet P-40, 10% glycerol, 1 mg/ml bovine serum albumin, 0.5 mm phenylmethylsulfonyl fluoride, and 1× Complete (Roche Molecular Biochemicals)) for 1 h at 4 °C. The beads were pelleted and washed five times with cold binding buffer. SDS-PAGE reducing buffer was added to the beads, samples were boiled for 5 min, and the eluted proteins were analyzed by SDS-PAGE. The gels were fixed and dried, and band intensities were quantified by phosphorimage analysis using the Bio-Rad GS 525 molecular imaging system.DISCUSSIONThe data presented here provide several lines of evidence that the PGC-1α·ERRα interaction represents a functional transcriptional complex involved in the regulation of cardiac and skeletal muscle metabolism. First, multiple independent ERRα clones were isolated as PGC-1α interacting proteins using a two-hybrid screen. The interaction of PGC-1α with ERRα and -γ isoforms was verified using in vitro binding assays. Specific binding domains were mapped within the PGC-1α·ERRα binding interface on both ERRα and PGC-1α, thereby indicating the specificity of the interaction. Interestingly, the ERRα binding domain within the PGC-1α molecule is unique compared with other transcription factor binding sites. Second, PGC-1α coactivated both ERRα and ERRγ isoforms in heterologous promoter-reporter assays. Third, involvement of PGC-1α·ERRα in regulating cellular metabolism is suggested by parallel tissue-specific and developmental expression profiles of ERR isoforms, PGC-1α, and mitochondrial FAO enzyme genes. A role for the PGC-1α·ERR complex in controlling mitochondrial FAO is further supported by the observation that PGC-1α is necessary for ERRα-mediated activation of the MCAD promoter.Mapping studies of the PGC-1α·ERRα binding interface revealed a novel LXXLL-type nuclear receptor binding motif on PGC-1α. Conserved LXXLL motifs within coactivator proteins have been shown to mediate AF-2 domain-dependent interactions with nuclear receptors. The Leu-rich motif adopts an α-helical conformation that fits into a hydrophobic binding pocket formed by several helices of the receptor LBD. PGC-1α contains three potential LXXLL motifs (L1–L3) although only one (L2) has been shown previously to play a major role in binding nuclear receptors. Our mutagenesis studies demonstrated that the interaction of ERRα with PGC-1α involves the L3 motif. Identification of an LXXLL motif as the ERRα binding site is consistent with the observation that the ERRα AF-2 domain is required for the interaction with PGC-1α. However, this is the first example in which the L3 motif of PGC-1α was found to mediate the interaction of PGC-1α with a nuclear receptor beyond a role as an accessory site for GR interaction with PGC-1α (12Knutti D. Kressler D. Kralli A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9713-9718Google Scholar). Our results indicate that the L3 motif functions as the primary site of interaction and that it is sufficient to bind ERRα. This proposed mechanism is distinct from most other NR interactions with PGC-1α, as demonstrated by the observation that the interaction of PGC-1α with ERα and HNF-4 required the L2 site.Although LXXLL defines the consensus signature motif through which coactivator proteins interact with nuclear receptors, numerous studies have established the importance of residues flanking the Leu-rich sequence in determining receptor selectivity and binding affinity (29Chang C.Y. Norris J.D. Gron H. Paige L.A. Hamilton P.T. Kenan D.J. Fowlkes D. McDonnell D.P. Mol. Cell. Biol. 1999; 19: 8226-8239Google Scholar, 30McInerney E.M. Rose D.W. Flynn S.E. Westin S. Mullin T.-M. Krones A. Inostroza J. Torchia J. Nolte R.T. Assa-Munt N. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1998; 12: 3357-3368Google Scholar). Chang et al. (29Chang C.Y. Norris J.D. Gron H. Paige L.A. Hamilton P.T. Kenan D.J. Fowlkes D. McDonnell D.P. Mol. Cell. Biol. 1999; 19: 8226-8239Google Scholar) recently defined three distinct classes of LXXLL domains based upon conserved flanking residues. According to this scheme, the PGC-1α L2 site is a class III binding site determined in part by the presence of Leu and Ser residues positioned immediately upstream of the first Leu of the LXXLL motif. The L3 motif, through which ERRα primarily interacts, appears to be an inverted LXXLL and, therefore, does not readily conform to this classification scheme. However, recent crystal structure studies performed with ERα demonstrated that the coactivator binding pocket of ERα could recognize an LXXφL motif within an NR box derived from the transcription intermediary factor 2 coactivator (31Pike A.C.W. Brzozowski A.M. Hubbard R.E. J. Steroid Biochem. Mol. Biol. 2000; 74: 261-268Google Scholar). Interestingly, despite the presence of a consensus LXXLL motif in the transcription intermediary factor 2 NR box, constraints placed by basic residues N-terminal to the α-helical motif shifted the binding site by one residue changing the recognition motif to LXXYL. The L3 motif of PGC-1α does, in fact, conform to an LXXYL consensus. Furthermore, a series of basic residues lie upstream of the L3 LXXYL motif that may contribute to its recognition by ERRα and ERRγ. Finally, consistent with these findings, the L1 motif (LLavL) does not match either of these consensus sequences and has not been identified as a high affinity binding site in any nuclear receptor AF-2-dependent interaction with PGC-1α (12Knutti D. Kressler D. Kralli A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9713-9718Google Scholar) (present study).The basis for the unique interaction of ERRα with PGC-1α compared with other L2-dependent PGC-1α nuclear receptor partners is unknown but is presumably related to structural differences within the nuclear receptor LBDs. The nuclear receptor interface with PGC-1α has not been precisely defined. However, p160 coactivator binding sites have been mapped for a number of receptors, including ERα, PPARγ, and retinoid X receptor α (31Pike A.C.W. Brzozowski A.M. Hubbard R.E. J. Steroid Biochem. Mol. Biol. 2000; 74: 261-268Google Scholar, 32Bourgult W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Google Scholar, 33Wisely R.T. Nolte G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Google Scholar). ERα interacts with NR boxes within transcription intermediary factor 2 via direct contacts between the LXXLL α-helix and ERα residues Glu-542, which resides within LBD helix 12, and Lys-362, which resides within helix 3. Both of these residues are conserved in ERR isoforms, suggesting that other structural differences within the LBD of ERRα and ERRγ account for their differential binding with PGC-1α. Modeling of the ERRα LBD based on homology with ERα reveals that 16 of 19 residues involved in ligand binding to ERα are either identical or conservative mismatches in ERRα (34Chen S. Zhou D. Yan C. Sherman M. J. Biol. Chem. 2001; 276: 28465-28470Google Scholar). Of the three distinct residues, Phe-329 was shown to be essential for the constitutively active conformation adopted by the LBD of ERRα (34Chen S. Zhou D. Yan C. Sherman M. J. Biol. Chem. 2001; 276: 28465-28470Google Scholar). Interestingly, the same residues are also distinct between ERRα and ERRγ. These findings suggest that, although these residues are not directly involved with coactivator binding, the amino acid differences may contribute to slight differences in LBD conformation among these receptors to influence the binding interface with PGC-1α. Lastly, a number of studies have described allosteric effects of DNA response elements on the conformation of bound receptors (35Wood J.R. Likhite V.S. Loven M.A. Nardulli A.M. Mol. Endocrinol. 2001; 15: 1114-1126Google Scholar, 36Starr D.B. Matsui W. Thomas J.R. Yamamoto K.R. Genes Dev. 1996; 10: 1271-1283Google Scholar, 37Wood J.R. Greene G.L. Nardulli A.M. Mol. Cell. Biol. 1998; 18: 1927-1934Google Scholar, 38Ikeda M. Wilcox E.C. Chin W.W. J. Biol. Chem. 1996; 271: 23096-23104Google Scholar). Recent findings suggest that the specificity of ERR isoforms for regulating particular target pr