The nuclear receptor coactivator PGC-1α exhibits modes of interaction with the estrogen receptor distinct from those of SRC-1

Estrogen receptor (ER) function is mediated by multi-domain co-regulator 29 proteins. A fluorescently labelled fragment of the human PGC-1 alpha coregulator (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-1a to ER. Anisotropy measurements revealed that the affinity of this PGC-1 alpha fragment for human ER alpha and P was fairly strong in the presence of estradiol (similar to 5 nM), and that unlike a similar fragment of SRC-1 (570-780), PGC-1(91-408) exhibited ligand-independent interactions with ER, particularly with ER beta (K-d similar to 30nM). Competition experiments of the complex between ERa and fluorescently labelled PGC-191-408 with unlabelled SRC-1(570-780) showed that PGC-1(91-408) 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 ER alpha, SRC-1570-780 and PGC-1(91-408). GST-pull-down experiments with deletion mutants of ER alpha revealed that the constitutive binding of PGC-1(91-408) 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-1 alpha 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. (c) 2005 Elsevier Ltd. All rights reserved.