Abstract
The localization of estrogen receptor (ER)β in mitochondria suggests ERβ-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERβ interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERβ interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERβ interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERβ with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.
Highlights
Estrogen receptor  and its domain interact with a host of brain mitochondrial and nuclear proteins
The presence of MnSOD activity in the gel assay in the mitochondrial fraction and absence of GAPDH and -actin by immunoblotting confirmed the purity of mitochondrial fraction (Fig. 1)
Timm44, a bonafide mitochondrial protein, was found in the nuclear fraction of mouse brain, whereas the nuclear pore complex 86 kDa protein was found in the mitochondrial fraction
Summary
Estrogen receptor  and its domain interact with a host of brain mitochondrial and nuclear proteins. The present findings provide evidence for the interaction of ER with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling. Estrogen receptor (ER), a ligand-activated transcription factor belonging to the nuclear receptor superfamily, has two main types: ER␣ and ER These receptors are localized intracellularly and following ligand binding, they regulate a large number of estrogen responsive genes [1, 2]. We report the identification of ER, transactivation domain (TAD) and ligand binding domain (LBD)-interacting proteins from the mitochondrial and nuclear extract of the mouse brain by pull-down and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) followed by analysis with mascot search engine and motif search software to determine the consensus binding motifs
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