Estrogen Receptor β and Its Domains Interact with Casein Kinase 2, Phosphokinase C, and N-Myristoylation Sites of Mitochondrial and Nuclear Proteins in Mouse Brain*

Background: Estrogen receptor β and its domain interact with a host of brain mitochondrial and nuclear proteins. Results: Estrogen receptor β interacting brain mitochondrial and nuclear proteins have consensus motifs. Conclusion: Estrogen receptor β interacts with casein kinase 2, phosphokinase C, and N-myristoylation sites present in mitochondrial and nuclear proteins. Significance: This might be useful to regulate estrogen-dependent gene regulation in brain for therapeutics. 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.

Estrogen receptor (ER), 2 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). They are also present in the plasma membrane and involved in many rapid, non-genomic effects via several signaling pathways (3,4). In addition, ER␤ is located in the mitochondria of estrogen target cells, including human breast cancer MCF-7 cells (5,6). ER interacts with a large number of factors such as steroid receptor coactivator (SRC)-1 (7), glucocorticoid receptor-interacting protein (GRIP)-1 (8), transcriptional intermediary factor (TIF)-2 (9), and others (10) that may activate (coactivators) or suppress (corepressors) the transcription of specific genes. Coactivators exhibit a region called helix-12 which includes NR boxes, each containing a LXXLL motif (where L is leucine and X represents any amino acid) (11), through which it interacts with ER as shown by functional in vitro and in vivo studies (12,13). Mapping studies with corepressors reveal that the receptor interaction and repression functions are separately located toward the C-terminal (14).
The corepressors namely silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT)and N-CoR have interaction motifs of short peptides comprising of 19 amino acids and 17 amino acids, respectively, with an internal signature motif (I/L)XX(I/V)I, which is sufficient for receptor interaction and ligand-induced dissociation. These motifs can adopt an amphipathic ␣-helical conformation, reminiscent of the signature motif LXXLL within coactivators (15). Mitochondrial localization of ER␤ helps to understand the mechanism(s) of neuronal death and develop neuroprotective drugs and therapeutic strategies that can delay or prevent Alzheimer disease and other chronic neurodegenerative conditions. In the brain, estrogens exhibit multiple effects that enhance or preserve functions during pathologic circumstances such as excitotoxic-ity, oxidative stress, and neurodegeneration. However, the ER␤ and its domain interacting proteins and their cellular functions are poorly understood in the brain, specifically in mitochondria. Here 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.

Cloning and Expression of ER␤, TAD, and LBD
Detailed procedures are discussed in supplemental Materials and Methods. Briefly, the same procedures were followed as reported earlier (16).

Purification of Recombinant Proteins
These are discussed in supplemental Materials and Methods. Briefly, the same procedures were followed as reported earlier (17).

Interaction of ER␤, TAD, and LBD with Mitochondrial and Nuclear Extract of Mouse Brain
Purification of Mitochondria and Preparation of Mitochondrial Extract-For mitochondrial purification (18), the mouse brain was dissected on ice and rapidly minced and homogenized at 4°C in isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/ml BSA, pH 7.4). Then the homogenate was centrifuged at 600 ϫ g for 5 min at 4°C. The supernatant was collected and centrifuged at 9,000 ϫ g for 8 min at 4°C. Thereafter the pellet was resuspended in isolation buffer containing heparin to release the mitochondria from synaptosome and again centrifuged at 9,000 ϫ g for 8 min at 4°C. Then the brownish mitochondrial pellet was resuspended in isolation buffer and spun at 9,000 ϫ g for 10 min at 4°C. The pellet containing mitochondria was resuspended in suspension medium (isolation buffer with 0.02% w/v heparin). Before preparing extract mitochondria were observed under microscope to avoid contamination (Fig. 1). The suspension was incubated with lysis buffer containing 20 mM HEPES (pH 7.6), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 0.4 mg/ml complete EDTA-free protease inhibitor (Roche Diagnostics) and stirred for 30 min on ice. Then it was centrifuged at 25,000 ϫ g for 40 min at 4°C. The supernatant containing the mitochondrial extract was checked quantitatively by the Bradford method and qualitatively by silver staining. The purity of mitochondrial extract was checked by in gel assay of MnSOD and immunoblotting using ␤-actin and GAPDH antibody. The detailed procedure is given in supplemental Materials and Methods.
Pull-down Assay-The detailed procedure is given in supplemental Materials and Methods.
Preparation of Nuclei and Nuclear Extract, Immunoblotting, Immunoprecipitation, and far-Western Blotting-For these techniques, the same procedure was followed as reported earlier (19). Nuclei were observed under a microscope (Fig. 1), and the purity of nuclear extract was checked by in-gel assay of MnSOD and immunoblotting by ␤-actin and GAPDH antibody. The detailed procedure is given in supplemental Materials and Methods.

MALDI-MS Analysis
The detailed procedure is given in supplemental Materials and Methods.

Identification of Putative Proteins
The detailed procedure is given in supplemental Materials and Methods.

Identification of Motifs
The detailed procedure is given in supplemental Materials and Methods. Briefly, the same procedure was followed as earlier reported (20).

Peptide Pull-down Assay
All the peptide sequences were purchased from GL Biochem Ltd, Shanghai, China. The peptide sequence from N to C ter- minus; namely for (casein kinase) CK2: RRREDEESDDEEEA, mutated CK2: RRREDEEPPALEEEA, (phosphokinase) PKC: CRFARKGSLRQKNV, mutated PKC: CRFARKGPPAQKNV, N-myristoylation: RCRMGGFLATSLSW, and mutated N-myristoylation: RCRMAAIPLASLSW were used for the peptide pull-down assay. All the peptide sequences were biotin tagged at the N terminus. For the interaction study, CK2, PKC, and N-myristoylation sites and their respective mutants were used. Briefly, 1 mg of each peptide was dissolved in 1 ml and aliquoted. Avidin beads were purchased from Sigma, suspended  in 400 l of 1ϫ PBS, and incubated with 100 g of biotinylated peptide overnight. Then it was washed three times with 1 ml of PBS to remove unbound peptides. Thereafter, 15 l of washed peptide bound beads and 5 g of purified ER␤, TAD, and LBD were incubated in 400 l of binding buffer for 4 h at 4°C. After incubation, the beads were washed with washing buffer three times. Then the beads-protein complex was resuspended in 40 l of Laemmli buffer and boiled at 100°C for 5 min. Finally, it was centrifuged at 10,000 ϫ g for 5 min, the supernatant was resolved by 7.5% SDS-PAGE, and the band was detected by silver staining.

RESULTS AND DISCUSSION
Before conducting experiments using mitochondrial and nuclear fractions, the purity of these fractions was checked. 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). Further, the absence of MnSOD activity by in-gel assay and the presence of ␤-actin and GAPDH by immunoblotting confirmed nuclear extract purity (Fig. 1).
Before performing the pull-down assay, we have analyzed the ER␤ and its domains interacting proteins using bioinformatics tools (STRING 8.0, PIP, BIND, and DIP) and literature-based studies (iHOP, NCBI, and ISI web of knowledge). This showed several interacting partners of ER␤, but the pull-down assay revealed relatively lower numbers of proteins, 16 in the range of 30 -172 kDa in mitochondria (Fig. 2) and 31 in the range of 31-203 kDa in the nuclei (Fig. 3). 3 Surprisingly 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. Timm 44 crosses through the nuclear pore and attaches to the translocator proteins of nuclear membrane (21). Similarly, Selvam et al. (22) identified some of the nuclear pore complex binding proteins in the mitochondrial extract of rat kidney. Two steps were used for identifying the proteins using mascot search engines. In the first step, all the peptide peaks including intense ions 842.933, 1476.33 1941, and 2384.7 in Fig. 1A, 1123.9 in Fig. 1C, and 1123.9 in Fig. 1D were used. It provided a list of proteins with various mascot scores and best matched unique peptide sequences. In the second step, these unique sequences were used to make identification more stringent with the higher mascot score. Detailed procedures have been provided in supplemental Materials and Methods. Also, one excel file showing procedure has been provided in the supplemental data. Thus, the proteins identified were more stringent with higher mascot score and collated to brain functions.

Interaction with Mitochondrial Proteins
ER␤-interacting Proteins-ER␤ showed interaction with six proteins of 152 kDa (prenylated protein-tyrosine phosphatase, peaks-36, unique peptides-11 and mascot score-149), 101 kDa (BC026557 similar to nucleoporin, peaks-29, unique peptides-11 and mascot score-153), 86 kDa (AK009187 similar to mitotic phosphoprotein, peaks-35, unique peptides-13 and mascot score-170), 71 kDa (phosphoinositol 3-phosphate-binding protein, peaks-26, unique peptides-22 and mascot score-190), 40 kDa (MKIAA1251 protein peaks-31, unique peptides-17 and mascot score-84), and 35 kDa (MUSACMHCA alpha cardiac myosin heavy chain peaks-31, unique peptides-14 and mascot score-56) ( Fig. 2A and supplemental Table S1A and Fig. S1, A-F). The 152 kDa protein matches to a receptor-like transmembrane protein and causes dynamic tyrosine phosphorylation and dephosphorylation at serine/threonine and participates in many aspects of cellular, physiological and pathological processes (23). The 86 kDa protein is a member of nucleoporins, which constitute the nuclear pore complex and interacts with transiently associated nuclear transport factors (24). The 71 kDa protein recruits cytosolic proteins to specific membrane compartment and acts as a spatial regulator of many cellular processes including endocytic trafficking and membrane fusion (25). The 40 kDa protein is involved in G-protein signaling and vesicle trafficking. Its mRNA in mouse brain has long 3Ј-UTR, which increases with evolutionary age and organism complexity (26). TAD-interacting Proteins-TAD showed interaction with four proteins of 172 kDa (Abhd4 protein, peaks-32, unique peptides-13, and mascot score-160), 58 kDa (aldehyde reductase, peaks-29, unique peptides-17, and mascot score-199), 50 kDa (survival of motor neuron, peaks-31, unique peptides-4, and mascot score-42) and 43 kDa (AIG1 family/TONB Box N terminus, peaks-36, unique peptides-20, and mascot score-158) ( Fig. 2B and supplemental Table S1B and Fig. S1, G-J). The 172 kDa protein is a lysophospholipase selective for N-acyl phosphatidylethanolamine (NAPE) and is involved in the biosynthesis of endocannabinoid anandamide by hydrolyzing the sn-1 and sn-2 acyl chains from NAPE generating glycerophospho-N-acyl ethanolamine (GP-NAE), an intermediate for N-acyl ethanolamine biosynthesis. It hydrolyzes substrates bearing saturated, monounsaturated, and polyunsaturated N-acyl chains. Its interaction with ER␤ suggests the role of estrogen in the regulation of the cannabinoid receptor and neuroprotection against cannabinoids (27). The 58 kDa protein is aldo-keto reductase, which catalyzes a number of redox transformations of glucose, steroids, glycosylation end products, lipid peroxidation products and environmental pollutants involved in biosynthesis, intermediary metabolism, and detoxification. It plays an important role in the detoxification of a large number of pharmaceuticals, drugs, and xenobiotics (28). The 50 kDa protein binds to small actin-binding protein and is involved in snRNP transport and nuclear RNA splicing (29).
LBD-interacting Proteins-LBD interacted with six proteins of 161 kDa (E130112J05 product protein, peaks-36, unique peptides-7, and mascot score-126), 103 kDa (Arhgap28 protein, peaks-33, unique peptides-24 and mascot score-188), 88 kDa (AK014612, f-box only protein, peaks-33, unique peptides-15, and mascot score-162), 73 kDa (LOC433520 protein, peaks-32, unique peptides-9, and mascot score-92), 41 kDa (kinesin-related microtuble-based motor protein, peaks-32, unique peptides-13, and mascot score-62) and 37 kDa (vinculin, peaks-32, unique peptides-20 and mascot score-136) ( Fig. 2C and supplemental Table S1C and Fig. S1, K-P). The 103 kDa protein is cytoskeletal and provides mechanical support to the plasma membrane and is involved in cell signaling, protein trafficking, control of cell proliferation, cell migration, adhesion, cytokinesis, and transcriptional activity (30). The 41 kDa is putatively identified as kinesins which are expressed in juvenile tissues including differentiated young neurons. Its expression is decreased considerably in adult mouse. It is involved in the transport of organelles through the axon, nucleotide-dependent binding to microtubules, microtubule-activated ATPase activity and microtubule plus-end-directed motility (31). The 37 kDa vinculin is a member of kinesin-like proteins (KLPs). It is involved in the localization of the mitotic spindle and ATP hydrolysis to move along microtubules in a unidirectional manner, transporting chromosomes or other microtubules toward either the plus or minus end of the microtubule polymer (32).

Interaction with Nuclear Proteins
ER␤-interacting Proteins-ER␤ interacted with seven putative proteins of 203, 140, 84, 43, 39, 33, and 30 kDa in mouse brain ( Fig. 3A and supplemental Table 2A and Fig. S2, A-F). The 140 kDa protein was identified as estrogen receptor associated protein (ERAP) 140 by immunoblotting, immunoprecipitation, and far-Western blot analysis (Fig. 4A), and the remaining proteins by MALDI-MS. The variation in MW determined by pull-down and MALDI-MS analysis was due to their multisubunit characteristics and post-translational modifications, especially glycosylation.
The 203 kDa protein matches putatively to MKIAA1251 (peaks-20, unique peptides-22 and mascot score-109) and Ugcgl2 (peaks-20, unique peptides-13, and mascot score-104). The 140 kDa protein is characterized as a conserved tissuespecific nuclear receptor coactivator with abundant expression in brain, exclusively in neurons. Its homologues are present in both invertebrates as well as vertebrates including human (33). We recently reported that mouse ER␤ interacts with estrogen receptor-associated protein (ERAP) 140 and shows variation in the levels of interaction and expression during aging of mouse brain (19). The 84 kDa protein matches to MMU10551(peaks-28, unique peptides-16 and mascot score-84), which is a member of the Ras superfamily and plays an important role in the regulation of L-type calcium channels and diverse cellular processes like cell growth, differentiation, cytoskeleton regulation, vesicular transport, cytokinesis, phagocytosis, pinocytosis, cell migration, morphogenesis, and axon guidance (34). The 43 kDa band matches to salivary androgen-binding protein (ABP) ␥ subunit fragment (peaks-25, unique peptides-7 and mascot score-63), which plays a role in sexual selection during brain development (35). The 39 kDa protein matches to adult male epididymis cDNA, RIKEN full-length enriched library, clone: 9230110G02 product: ATP/GTP-binding site motif A (P-loop)/ zinc finger (peaks-25, unique peptides-19 and mascot score-183) (36). The 33 kDa protein matches to MMFXR1H9 (peaks-29, unique peptides-17, and mascot score-175), which is present in the brain and shows estrogen dependent expression and regulation (37). The 30 kDa protein matches to adult male small intestine cDNA clone product, RIKEN full-length enriched library, clone:2010015J01 product:weakly similar to Arp2/3 complex 16 kDa subunit (P16-ARC, actin-related protein 2/3 complex subunit 5, peaks-18, unique peptides-12, and mascot score-156) which is present in the brain of fetal down syndrome patient and is responsible for neuronal and glial migration during brain development (38).
TAD-interacting Proteins-TAD interacted with ten proteins of 160, 100, 98, 85, 69, 61, 53, 45, 34, and 31 kDa (Fig. 3B and supplemental Table S2B and Fig. S2, G-O). The 160 kDa protein was identified as amplified in breast cancer (AIB) 1 by immunoblotting, immunoprecipitation, and far-Western blot analysis (Fig. 4B). AIB1 is a member of the nuclear coactivator (NCoA-3) and p160 steroid receptor co-activator (SRC) family, which includes SRC-1 and TIF-2 and plays a role in breast cancer and enhances angiogenesis and neurogenesis (39). We recently reported that mouse ER␤ interacts with amplified in breast cancer (AIB) 1 (38) and showed variation in the levels of interaction and expression during aging of mouse brain. The 100 kDa protein is probably an iron-binding protein P97 (peaks-31, unique peptides-8, and mascot score-127), but its physiological role is not clear in brain. Originally, it was discov-ered in melanoma cells, but now it is known to be expressed at varying levels in several kinds of tumors. In normal tissues, it is expressed at high levels in fetal gut, adult sweat gland, and probably brain capillaries. P97 is structurally homologous to transferrin and is shown to bind to iron and zinc. It is reported as a marker in Alzheimer patients, which show its elevated level in serum (40). The 98 kDa protein is identified as an adult inner ear protein (peaks-20, unique peptides-6, and mascot score-113), which is a member of Nkx5-1 family and has various roles in inner ear and hindbrain development (42). The 85 kDa protein matches to MKIAA 0670 (peaks-24, unique peptides-23, and mascot score-139), which is a member of KIAA family with unknown biological function (43). The 69 kDa protein has been identified as 18th day whole body protein (peaks-26, unique peptides-9, and mascot score-112), and acts as a homeodomain transcription factor in brain. The 61 kDa protein is a ankyrin protein (peaks-31, unique peptides-19 and mascot score-187), expressed in brain and plays a vital role in energy homeostasis (44). The 53 kDa protein is identified as bone marrow macrophage tRNA synthetase (peaks-35, unique peptides-22, and mascot score-192). The 45 kDa protein matches to melanocyte protein (peaks-26, unique peptides-22, and mascot score-207) known for induction of melanocyte-stimulating hormone and its related function in neurons (45). The 34 kDa protein  Table S2C and Fig. S2, P-X). Among them 140, 138, 60, 45, and 43 kDa were identified as ERAP 140, TrkA, Src, pCREB, and cAMP response element binding protein (CREB), respectively, by immunoblotting, immunoprecipitation, and far-Western blot analysis (Fig. 4C).
The 179 kDa protein has been identified as a molydopterin converting factor subunit 2 containing protein (peaks-24, unique peptides-12, and mascot score-163), and functions as a cofactor in metabolic activity (46). The 138 kDa protein is Trk A, which is known for the autophosphorylation and recruitment of signaling molecules and activates a receptor-specific intracellular signaling pathway that relays information to nucleus and other intracellular compartments (47). The 136 kDa protein is identified as 2nd day oviduct protein (peaks-21, unique peptides-12 and mascot score-122), and has no well known function. The 115 kDa protein is Tim 44 (peaks-18, unique peptides-15, and mascot score-137), which is responsible for the transportation of proteins to membrane and suborganelles including mitochondria. It controls the oxidative phosphorylation and takes part in the respiratory chain enzyme kinetics (48). The 99 kDa protein is heparan sulfate sulfotransferase isoform 6 (peaks-29, unique peptides-16, and mascot score-182), and is reported in the brain. The 88 kDa protein matches to tomoregulin (peaks-16, unique peptides-13, and mascot score-140), and is a transmembrane protein predominantly expressed in the brain. The 69 kDa protein has been identified as senataxin (peaks-23, unique peptides-21, and mascot score-70), which causes a rare juvenile-onset form of amyotrophic lateral sclerosis (ALS). The 58 kDa matches to SRY protein (peaks-24, unique peptides-14, and mascot score-127), known as the key sex-determining protein that directs gonads to develop as testes rather than ovaries. The 60 kDa protein is identified as Src which belongs to a family of highly homologous proteins with protein tyrosine kinase (PTK) activity and resides in the cytoplasm and perinuclear space. Recent reports suggest that Src is also located at the plasma membrane and several subcellular compartments including the nucleus (49) and involves in signal transduction. The 49 kDa protein has been identified as a member of KIAA gene family (peaks-23,

ER␤-interacting Mitochondrial and Nuclear Proteins in Brain
unique peptides-13, and mascot score-118), and its role in brain function is not clear (41). The 45 kDa and 43 kDa proteins have been identified as pCREB and CREB, respectively. They are well known transcription factors involved in multiple brain functions. Moreover, 42 kDa protein is identified as alanine region protein (peaks-29, unique peptides-9, and mascot score-123), and is known for its role in signal transduction in the brain (50).

Identification of Consensus Motifs
As all the mitochondrial and nuclear proteins interacted with ER␤ and its domains, we assume that these proteins have a common consensus binding motifs. Therefore, all the interacting proteins were analyzed by motif scan, and interestingly they were found to contain consensus motifs CK2, PKC phosphorylation, and N-myristoylation sites (Fig. 5 and Fig. 6). This was also confirmed by peptide pull-down assay using specific biotinylated CK2, PKC, and N-myristoylation peptides. CK2, PKC phosphorylation and N-myristoylation sites showed interaction with ER␤, TAD and LBD whereas no interaction was observed with their respective mutants (Fig. 7). The gels show the binding difference between normal and mutated peptides with ER␤ and its domains. In string 8.0, peptide sequences did not show any match with putative proteins. So it is likely that these are bacterial proteins contaminations. Both CK2 and PKC phosphorylation sites start with serine or threonine residue, and phosphorylation at these sites is responsible for the interaction of all the putatively identified proteins with ER␤ result-ing in the downstream functions, namely neuronal architecture, migration of neuronal and glial cells during development and cell proliferation (51). Further, N-myristoylation is involved in the anchoring of proteins and their targeting to cell membrane and plays a critical role in cell signaling, apoptosis and extracellular protein export. The myristoyl-CoA and N-myristolyltransferase (NMT) recognize the sequence motif of myristate site at N-terminal glycine residues. Apart from anchoring, myristoylation is also involved in the docking of Ca 2ϩ (52).
As protein interactions are transient, our proteomic approach precisely analyzes the interacting proteins by pulldown assay followed by MALDI-MS and identifies their motifs by motif scan software. The identification of ER␤-interacting proteins in brain mitochondria indicates the importance of ER␤ in the regulation of mitochondrial gene expression. This is contradictory to an earlier observation in which ER␤ was reported as a poor transcription factor (53,54). The present study is of great significance as it advances our knowledge of nuclear receptor and its interaction and functional analysis in the brain mitochondria. The putatively identified proteins are involved in cellular signaling in mitochondria. The reduced level of putatively identified survival motor neuron (SMN) protein causes spinal muscular atrophy. The CK2, PKC, and N-myristoylation sites have been identified for the first time as consensus motifs in all ER␤ interacting proteins in the brain mitochondria. This raises the possibility to regulate the estro- gen dependent gene expression through ER␤ in health and disease (10).
Taken together, our studies showed that ER␤ and its domains interact with the consensus CK2, PKC phosphorylation, and N-myristoylation sites of specific mitochondrial as well as nuclear proteins. Evidences from literature suggest that these interacting proteins are involved in neuronal architecture, cytoskeleton integrity, signal transduction, and cell proliferation. The identification of consensus binding motifs in ER␤interacting proteins of the brain mitochondria represents an essential step toward designing selective ER modulators for understanding estrogen-mediated signaling.