The Nuclear Receptor Coactivator PGC-1α Exhibits Modes of Interaction with the Estrogen Receptor Distinct From those of SRC-1 Original Research Article

Estrogen receptor (ER) function is mediated by multi-domain co-regulator proteins. A fluorescently labelled fragment of the human PGC-1α co-regulator (residues 91–408) bearing the two motifs most strongly implicated in interactions with nuclear receptors (NR box2 and NR box3), was used to characterize in vitro binding of PGC-1α to ER. Anisotropy measurements revealed that the affinity of this PGC-1α fragment for human ERα and β was fairly strong in the presence of estradiol (∼5 nM), and that unlike a similar fragment of SRC-1 (570–780), PGC-191–408 exhibited ligand-independent interactions with ER, particularly with ERβ (Kd∼30 nM). Competition experiments of the complex between ERα and fluorescently labelled PGC-191–408 with unlabelled SRC-1570–780 showed that PGC-191–408 was an efficient competitor of SRC-1570–780, while the inverse was not true, underscoring their distinct modes of binding. The anisotropy data provide strong evidence for a ternary complex between ERα, SRC-1570–780 and PGC-191–408. GST-pull-down experiments with deletion mutants of ERα revealed that the constitutive binding of PGC-191–408 requires the presence of the linker domain between the DNA binding and ligand binding domains (DBD and LBD). Homology modeling studies of the different regions of full length PGC-1α confirmed the lack of compact tertiary structure of the N-terminal region bearing the NR box motifs, and suggested a slightly different mode of interaction compared to the NR box motifs of SRC-1. They also provided reasonable structural models for the coiled-coil dimerization motif at residues 633–675, as well as the C-terminal putative RNA binding domain, raising important questions concerning the stoichiometry of its complex with the nuclear receptors.