How does PELP1 perform these various cellular roles? PELP1 neither binds

How does PELP1 perform these various cellular roles? PELP1 neither binds specific sequences on the DNA nor is involved in signal transduction at the plasma membrane not has any known enzymatic activity. The explanation for the ascribed roles of PELP1 may lie in its extensive social network with other protein partners. PELP1 has multiple structural motifs that enable its direct interaction with a number of key proteins (Figure 1) (10); by functioning as a scaffold, PELP1 enables cross talk between its interacting proteins (11,C14). The interactome of PELP1 is extensive and includes SRs, chromatin-modifying enzymes, cellular kinases, splicing factors, and transcriptional regulators (Figure 2). PELP1 likely indirectly but critically influences cell signaling and cellular processes. This review critically evaluates the molecular basis of the role of PELP1 in hormonal signaling and cancer. Open in a separate window Figure 1. Primary domain structure of PELP1.PELP1 has 11 LXXLL motifs for interactions with SRs, 2 proline-rich regions and PXXP domains for interactions with signaling proteins, a glutamic acid-rich carboxy region for interactions with histones. PELP1 also contains 2 cysteine-rich regions, which could possibly form zinc fingers for protein interactions, a nuclear localization signal (NLS), 3 nucleolar localization domains (NuLD), and a RIX1 domain. Open in another window Figure 2. PELP1 being a scaffolding proteins.PELP1 interacts with SRs such as for example AR, ER, and PR through its LXXLL theme, SR coregulator protein such as for example FHL2, p300, CBP, and Src, and chromatin-modifying protein such as for example KDM1, coactivator-associated arginine methyltransferase 1 (CARM1), and histones through its glutamic acid-rich region. The interactome of PELP1 allows cross chat between these proteins and with the DNA. Id of PELP1 PELP1 was originally defined as a Src homology 2 (Sh2)-binding proteins within a glutathione-S-transferase-pull straight down screening research (15). It was purified later, peptide sequenced (16), and a cDNA clone isolated from a HeLa cDNA collection (1). The causing 160-kDa proteins was found to become abundant with proline, glutamic leucine and acid, which constitute about 40% of the full total proteins and donate to the eponymous name. Because overexpression of PELP1 improved estradiol-dependent transcriptional activation in the estrogen receptor (ER) response component, PELP1 was specified being a coactivator of ER (17). Primary Structure as well as the Interactome of PELP1 PELP1 is a 160-kDa scaffolding proteins with several motifs within coregulators (uniprot commonly.org/uniprot/Q8IZL8). The principal structure presents significant signs to its putative function. A domains scan using an internet device (http://scansite.mit.edu) revealed several protein-interacting domains on PELP1 (Amount 1). Included in this is a solid nuclear localization series (focused at amino acidity 640) (1) and 3 nucleolar localization domains (proteins 74C152, 423C489, and 569C639). Unsurprisingly, PELP1 is basically nuclear generally in most studies (1). PELP1 contains 10 distinct LXXLL motifs (L is leucine, x is any residue, also called nuclear receptor container), which enable its connections with SRs (18). Connections of PELP1 continues to be noted with ER, androgen receptor (AR) (11, 19), estrogen-related receptor- (20), and retinoid X receptor (RXR) (4) and could be improved with ligand binding towards the SR (1, 14). Each LXXLL theme within PELP1 provides exclusive flanking sequences, which might dictate a differential affinity for every SR (21). For instance, ER preferentially interacts using the 4th and 5th LXXLL motifs on PELP1 (21). Furthermore, PELP1 might user interface with SRs through motifs apart from the LXXLL theme, for instance, PELP1 interacts with glucocorticoid receptor (GR) through its proline- and glutamic acid-rich locations (12). PELP1 has several potential Src-binding sites also, which take into account its preliminary isolation within a Src-pull straight down. Mechanistic studies show a proline-rich area in the N terminus of PELP1 (PxxP, where P is normally a proline, x is normally any residue) interacts using the SH3 theme of c-Src. Furthermore, the SH2 motifs of PELP1 represent high-affinity connections sites for c-Src. The close closeness of nuclear receptor containers and c-Src-binding sites on PELP1 may enable mix speak between c-Src and SRs by developing complexes (19, 21). Such complexes of AR, Src, and PELP1 had been observed in androgen-dependent LNCaP cells upon androgenic arousal and constitutively within androgen-independent LNCaP cells (19). PELP1 doesn’t have an affinity for distinct DNA sequences but interacts using the chromatin. The extremely acidic glutamic acid-rich domains inside the C terminus of PELP1 allows its connections with simple chromatin and recruitment of histone-modifying enzymes (22, 23). Hence, PELP1 may possess a functional function on chromatin redecorating by displacing histone H1 (22). Finally, dispersed throughout PELP1 are extensive cognate phosphorylation sites for potential regulation of its activity simply by kinases. PELP1 acts as a substrate for most kinases, including proteins kinase A (24) and cyclin-dependent kinase (CDK) (25). The importance as well as the function of PELP1 phosphorylation in mobile processes aren’t known. Changed Expression of PELP1 in Cancer The expression of PELP1 is deregulated in a number of hormonal cancers, including breast (1, 26, 27), ovarian (28), endometrial (29, 30), and prostate (6) cancers. Duplicate number modifications of PELP1 gene have already been noted in a part of these malignancies (over 2% of prostate malignancies, 1.5% of ovarian cancers, and 1% of breast cancers, current analysis from the Cancer Genome Atlas databases, May 2015). In breasts tumors, PELP1 appearance is apparently correlated with tumor quality and invasiveness favorably, and inversely correlated with disease-free survival (27, 31, 32). Of be aware, high PELP1 appearance and low ER, progesterone receptor (PR), and AR appearance were seen in intrusive breasts malignancies (27). Changed subcellular localization of PELP1 continues to be seen in a subset of breasts and endometrial cancers (26, 30). PELP1 appearance was also associated with high tumor quality in astrocytic human brain tumors (33). Although PELP1 appearance was higher in digestive tract and salivary gland tumors weighed against normal tissue, no correlation could possibly be set up to tumor quality or hostility (34,C36). Mutations in PELP1 have already been detected in a variety of malignancies but not sufficiently studied. Function in Genomic SR Signaling Preliminary characterization revealed a good amount of PELP1 in the mammary epithelium, where ER is normally predominant (1). PELP1 was proven a coactivator of estradiol-induced ER-driven gene appearance then. PELP1 can boost or repress SR transactivation based on mobile framework and ligand focus (12). For instance, in A549 cells that express significant endogenous GR, PELP1 inhibited GR transactivation. On the other hand, in HEK293 cells that RSL3 supplier express suprisingly low degrees of endogenous GR, PELP1 improved ligand-dependent GR transactivation. Biphasic behavior of exogenously portrayed signaling substances (ie, frequently when overexpressed) is normally indicative of scaffold activity, where stoichiometric concentrations from the interacting elements that take part within a proteins complicated might assist in maximal indication result, whereas an excessive amount of a specific player (ie, in this full case, the scaffold itself) may hinder signal result via sequestration of just one 1 of even more of the interacting elements (37). Suspected scaffold activity could be analyzed by physiologic manipulation from the degrees of 1 proteins (ie, changing total PELP1 amounts using little interfering RNA (siRNA) or brief hairpin RNA knockdown or exogenous PELP1 overexpression) while preserving the same degrees of its interacting companions. PELP1 interaction with SRs is mediated through its multiple LXXLL domains, and the sequences flanking each LXXLL domain name mediate its specificity for individual SRs. PELP1 may also interact with SRs through non-LXXLL domains For example, exogenous overexpression of fragments of GR in HEK293 cells revealed that PELP1 interacts with both the amino and the C termini of GR. PELP1 was able to regulate both activation function 1 and activation function 2 transactivation functions of GR through these differential interactions (12). The role of PELP1 in genomic signaling mediated by multiple SRs has been well characterized. For example, PELP1 cooperates with the orphan SR, estrogen-related receptor- to increase aromatase gene expression (20). Knockdown of PELP1 affects the genomic signaling mediated by several SRs, with multiple mechanisms proposed. PELP1 may enable SR genomic signaling via its interactions with traditional SR-transcriptional coactivators, such as and the closely related CREB binding protein (CBP) (Physique 3A) (38). PELP1 may recruit both SRs and their specific coactivators, thereby decreasing the entropy of such interactions within a cell via forced proximity. PELP1 may also stabilize SR-coregulator complexes that normally interact weakly in the absence of PELP1 (Physique 3A). PELP1 may also induce chromatin remodeling at ER target gene promoters by displacing histone H1 (22). PELP1 mediates methyl modifications of histone H3 at gene promoters via interactions with histone demethylase KDM1 (lysine-specific histone demethylase 1A) (23) or coactivator-associated arginine methyltransferase 1 (CARM1) (Physique 3A) (39). PELP1 may enable SRs to interact with some coregulators, even the SRs and coregulators cannot directly interact. For example, PELP1 aids the AR-coregulator four-and-a-half lens intrinsic membrane protein 2 (FHL2) that lacks motifs for direct interactions with AR by binding both AR and FHL2 at the N-terminal region (1C600 amino acids); the close proximity of these proteins on PELP1 enables the coregulator and AR to functionally interact without a lead physical conversation (11). PELP1 further aids in FHL2 transactivation function by binding other proteins through its SH3, PDZ, and WW motifs at its C-terminal end (Physique 3B). Thus, PELP1 may serve as a platform or node for the recruitment and business of transcription complexes. Open in a separate window Figure 3. Role of PELP1 in SR signaling.SR bound to PELP1 is translocated into the nucleus, where it binds chromatin. PELP1 facilitates transcription of SR-regulated genes by recruiting coregulators p300, CBP, and HAT to the chromatin that displace histone H1, and by interacting with KDM1 that causes methyl modifications on histone H3. In the absence of ligand, PELP1 may couple an unliganded SR to a liganded SR and activate genomic signaling. In the cytoplasm, PELP1 interacts with both Src and SRs. However, the role of PELP1 in nongenomic signaling needs to be robustly validated. Role in Nongenomic SR Signaling Although PELP1 is predominantly localized in the nucleus, its localization to the cytoplasm has been observed in a subset of breast tumors (26, 40). In the cytoplasm, PELP1 may couple SRs with kinase signaling cascades, as shown for AR, which enables conversation with signaling proteins such as G proteins (14) and Src (19). Mutations of the PxxP domains on PELP1 resulted in the loss of ER-Src conversation and estrogen-induced MAPK activation (13). Overexpression of cytoplasmic PELP1 resulted in the quick induction of MAPK and AKT signaling pathways in a hormone-independent manner, which in turn phosphorylated ER (Physique 3) (26, 41). Complexes of AR, PELP1, and Src have been observed in LNCaP cells; however, their role in Src signaling has not been validated (19). The role for PELP1 in nongenomic SR signaling in mammalian cells has been proposed but requires strong validation and remains unproven. Role in SR Cross Talk PELP1 functions as a bridge for SRs, allowing the activation of just one 1 SR to activate another SR, in the lack of the ligand for the next SR also. For instance, in prostate tumor cells, in the lack of androgens, PELP1 facilitates estradiol binding to ER towards the unliganded AR and activates AR signaling in these cells (Body 3) (6). Knockdown of PELP1 abrogated the coupling of liganded ER sign to unliganded AR (6). Likewise, in breast cancers cells, PELP1 facilitated combination chat between PR and ER, in the lack of the PR ligand progestin (8). Gene appearance analyses indicated the fact that relationship between PR, PELP1, and ER allowed the activation of the subset of ER-target genes (8). Jointly, these data indicated that PELP1 might enable a liganded SR to impact the experience of another unliganded SR or vice versa. This acquiring provides significant implications for level of resistance pathways in endocrine malignancies, where in fact the ligands of particular SRs are targeted. Function in Chromatin Modification PELP1 participates being a transcriptional coactivator/corepressor of non-nuclear receptors activator proteins 1, nuclear aspect B, ternary complicated aspect/serum response aspect (2). The glutamic acid-rich area of PELP1 allows its relationship using the chromatin and relationship with simple histones (Body 2). Although PELP1 will not enhance the histones straight, its recruitment of histone modifiers allows histone modification, for instance, the leucine-rich N-terminal area of PELP1 recruits histone deacetylase 2 towards the chromatin, which, subsequently, deacetylateslysine residues on primary histones (2). PELP1 binds towards the hypoacetylated histones and protects them from acetylation after that, hindering chromatin redecorating and influencing gene transcription thereby. Biological Jobs of PELP1 PELP1 might play critical jobs in a number of biological procedures, including proliferation, cell routine rules, and apoptosis. Many of these suggested roles have already been inferred from overexpression or knockdown tests. Nevertheless, because PELP1 is apparently critical for mobile success, siRNA-mediated knockdowns of PELP1 manifestation rarely surpasses 80% of proteins level and significant PELP1 manifestation level can be recognized (6, 14, 42). To your knowledge, there is absolutely no solitary cell line that will not communicate PELP1. At this right time, it really is unclear whether cells genetically manufactured never to communicate PELP1 using clustered frequently interspaced brief palindromic repeats or transcription activator-like effector nuclease methods will survive. The real role and extent of PELP1 in these cellular processes is thus not evaluable. Proliferation PELP1 has been proven to make a difference for both estrogen-independent and estrogen-induced proliferation in breasts tumor cells (7, 20, 28, 29, 43, 44). In ER-positive breasts cancers, PELP1 manifestation correlated with tumor size and mitotic count number (27). Knockdown of PELP1 reduced development of both ER-negative and ER-positive breasts and ovarian tumor cells (7, 28, 43). Overexpression of PELP1 in MCF7 cells improved its estradiol-dependent and estradiol-independent proliferation and induced mobile change (44). PELP1 may enhance proliferation of breasts tumor cells by raising regional estrogen synthesis (20, 45). In MCF7 cells, through the Src and phosphatidylinositide 3-kinases pathways, PELP1 improved aromatase expression, which may regulate estrogen amounts (20). Additionally, PELP1 mediates mix chat between ER and PR, which sensitizes cells to estradiol and IGF-1 (8). Collectively, these data support the oncogenic potential of PELP1 when dysregulated by overexpression and/or mislocalization. Cell Routine Regulation PELP1 has been proven to bind the critical cell routine change, the retinoblastoma proteins (pRb) (46). PELP1 actuates the phosphorylation of pRb at Ser807 and Ser811 (46). PELP1 itself can be a focus on for cyclin and CDKs D1, which is involved with pRb phosphorylation and activation (46, 47). Further proof for its part in the cell routine originates from oocytes through its discussion with membrane G protein, particularly G (14). Knockdown of PELP1 in these oocytes reduced G-signaling, overcame the meiotic arrest, improved maturation of oocytes and triggered the MAPK pathway. Apoptosis In response to stressors, such as for example chemotherapeutic drugs and ionizing radiation, DNA damage response kinases phosphorylate PELP1 at Ser1033 (48). Phosphorylated PELP1 binds tumor suppressor p53 and modulates p53 acetylation and phosphorylation areas, which affects its function in DNA harm response. PELP1 knockdown leads to faulty p53 signaling in response to stressors and impacts stress-induced apoptosis. Overexpression of PELP1 induces apoptosis through caspase-mediated cleavage from the DNA fix enzyme, poly ADP ribose polymerase, in response to TNF- (40). Additionally, PELP1 interacts with both RXR homodimers and RXR-peroxisome proliferator-activated receptor heterodimers and induces apoptotic activity (4). Ribosome Biogenesis PELP1 contains an RIX1 domains and sequences for nucleolar localization and exists in the nucleolus during S and G2 stages from the cell routine, when ribosomal transcription occurs (3). Nucleolar localization of PELP1 would depend on cell routine progression and could be governed by phosphorylation by CDK. PELP1 also interacts with RSL3 supplier 28S rRNA precursors as well as the 28S rRNA maturation aspect, LAS1-like protein, and could regulate bigger subunit maturation (49, 50). Tumor Metastasis and Migration PELP1 is expressed in every breasts cancer tumor cells highly. However, quantitative appearance is normally higher in metastatic breasts cancer tumor than in node-negative tumors. Knockdown of PELP1 changed many epithelial to mesenchymal changeover markers, including the ones that impact cell adhesion, migration, motility, and proliferation, and it is connected with a lack of migration potential of breasts cancer tumor cells, both in vitro and in mice versions (7). PELP1 also modulates microRNAs (miR-200 and miR-141) (51) involved with metastasis, by recruiting histone-modifying enzymes at their promoters possibly. Function in Therapy Resistance The role of PELP1 in several cellular processes in both ER-positive and ER-negative breast cancer cells in both hormone-dependent and hormone-independent pathways may enable a central role in therapy resistance. For instance, PELP1 elevated ER powered gene appearance in MCF7 cells in response to either epidermal development tamoxifen or aspect, also in the lack of estrogen (24). Mutational analyses uncovered that EGF and tamoxifen induced phosphorylation of PELP1 at 3 serine residues (S350, S415, and S613) by proteins kinase A, which turned on ER-mediated genomic signaling. PELP1 localization in the cytoplasm led to poor response to tamoxifen in transgenic mouse versions (31). The power of PELP1 to bridge SRs with nongenomic signaling mediated through players like Src and PI3 kinases may induce therapy level of resistance (9). Additionally, through its function in SR combination talk, PELP1 might activate ER signaling, in the lack of estrogen or progesterone ligand (9 also, 26). PELP1-mediated mix speak of PR-B with ER was discovered to activate a subset of ER focus on genes connected with luminal-B and success phenotype and could be an root mechanism of level of resistance to hormone therapy and tamoxifen in breasts cancer (8). Very similar processes could be very important to various other hormone-dependent or influenced cancers such as for example ovarian and breast cancers hormonally. Targeting PELP1 The involvement of PELP1 in a number of cellular processes and its own deregulation in hormonal cancers produce it attractive being a therapeutic target. Nevertheless, any agent concentrating on PELP1 should be examined for toxicity, as multiple SR-dependent and SR-independent pathways may be involved. A perfect agent would focus on PELP1 in tumor cells selectively. To time, nanoliposomal formulations of siRNA against PELP1 possess successfully been used to diminish tumor development and metastatic tumor nodules in ovarian xenograft tumor choices (42). Alternatively, concentrating on PELP1-combined nongenomic signaling pathways, including via the Src inhibitor, dasatinib, could be effective against PELP1-mediated hormone resistant tumors (ie, because of its ability to stop ER-PELP1-Src axis) (9). Recently, we confirmed the usage of peptidomimetics to inhibit prostate tumor cell proliferation simply by particularly blocking PELP1 connections with AR (52). The peptidomimetics had been designed to imitate the user interface between AR and PELP1 and obstructed the relationship between AR and PELP1. Significantly, these peptidomimetics usually do not influence PELP1 protein appearance or its relationship with other proteins companions. Because PELP1 is apparently central to AR function, administration from the peptidomimetic obstructed AR-driven signaling and AR-driven proliferation in these tumor cells both in vitro so that as xenografts in vivo. Significantly, these peptidomimetics didn’t show any proof toxicity in mice on the healing doses. These outcomes evince the chance of using peptidomimetics as a technique to focus on PELP1 without associated toxicity. Further, particular PELP1-protein interactions could be targeted with peptidomimetics made to block either specific subsets or interactions of interactions. Conclusions and Potential Directions There is installation proof that PELP1 has key roles in a number of biological processes and its own deregulation leads towards the advancement of malignancies. PELP1 seems to mediate these features by offering a scaffold that provides critical protein companions jointly for heightened signaling and transcriptional activity. PELP1 is basically appears and nuclear to connect to chromatin and modulate histones through its recruitment of histone-modifying enzymes. In dependent cancers hormonally, PELP1 is apparently crucial for both hormone-independent and hormone-dependent actions of multiple SRs. The function of PELP1 is apparently largely reliant on its cultural capability to interact with a big multitude of protein. PELP1 features may be both context-dependent and cell-line particular, dependant on its specific interactome. Even though the function of PELP1 in SR-mediated genomic signaling continues to be widely validated, its role in SR-driven nongenomic signaling remains unproven and controversial. The central scaffolding function of PELP1 helps it be a nice-looking target and selective targeting of specific interactions within the social network of PELP1 may be possible with peptidomimetics. Acknowledgments Funding was provided by the Congressionally Directed Medical Research Programs (W81XWH-12-0288 and W81XH-13-2-0093), the Cancer Prevention Research Institute of Texas (DP150096), and the Mimi and John Cole Foundation. Disclosure Summary: The authors have nothing to disclose. Funding Statement Funding was provided by the Congressionally Directed Medical Research Programs (W81XWH-12-0288 and W81XH-13-2-0093), the Cancer Prevention Research Institute of Texas (DP150096), and the Mimi and John Cole Foundation. Footnotes Abbreviations: ARandrogen receptorCBPCREB binding proteinCDKcyclin-dependent kinaseERestrogen receptorFHL2four-and-a-half lens intrinsic membrane protein 2GRglucocorticoid receptorKDM1Alysine-specific histone demethylase 1APELP1proline, glutamic acid, and leucine-rich protein 1PRprogesterone receptorpRbretinoblastoma proteinRXRretinoid X receptorSh2Src homology 2siRNAsmall interfering RNASRsteroid receptor.. cross talk between its interacting proteins (11,C14). The interactome of PELP1 is extensive and includes SRs, chromatin-modifying enzymes, cellular kinases, splicing factors, and transcriptional regulators (Figure 2). PELP1 likely indirectly but critically influences cell signaling and cellular processes. This review critically evaluates the molecular basis of the role of PELP1 in hormonal signaling and cancer. Open in a separate window Figure 1. Primary domain structure of PELP1.PELP1 has 11 LXXLL motifs for interactions with SRs, 2 proline-rich regions and PXXP domains for interactions with signaling proteins, a glutamic acid-rich carboxy region for interactions with histones. PELP1 also contains 2 cysteine-rich regions, which could possibly Mouse monoclonal to CD10.COCL reacts with CD10, 100 kDa common acute lymphoblastic leukemia antigen (CALLA), which is expressed on lymphoid precursors, germinal center B cells, and peripheral blood granulocytes. CD10 is a regulator of B cell growth and proliferation. CD10 is used in conjunction with other reagents in the phenotyping of leukemia form zinc fingers for protein interactions, a nuclear localization signal (NLS), 3 nucleolar localization domains (NuLD), and a RIX1 domain. Open in a separate window Figure 2. PELP1 as a scaffolding protein.PELP1 interacts with SRs such as AR, ER, and PR through its LXXLL motif, SR coregulator proteins such as FHL2, p300, CBP, and Src, and chromatin-modifying proteins such as KDM1, coactivator-associated arginine methyltransferase 1 (CARM1), and histones through its glutamic acid-rich region. The interactome of PELP1 enables cross talk between these proteins and with the DNA. Identification of PELP1 PELP1 was originally identified as a Src homology 2 (Sh2)-binding protein in a glutathione-S-transferase-pull down screening study (15). It was later purified, peptide sequenced (16), and a cDNA clone isolated from a HeLa cDNA library (1). The resulting 160-kDa protein was found to be rich in proline, glutamic acid and leucine, which constitute about 40% of the total protein and contribute to the eponymous name. Because overexpression of PELP1 enhanced estradiol-dependent transcriptional activation from the estrogen RSL3 supplier receptor (ER) response element, PELP1 was designated as a coactivator of ER (17). Primary Structure and the Interactome of PELP1 PELP1 is a 160-kDa scaffolding protein with several motifs commonly found in coregulators (uniprot.org/uniprot/Q8IZL8). The primary structure offers significant clues to its putative function. A domain scan using an online tool (http://scansite.mit.edu) revealed several protein-interacting domains on PELP1 (Figure 1). Among them is a strong nuclear localization sequence (centered at amino acid 640) (1) and 3 nucleolar localization domains (amino acids 74C152, 423C489, and 569C639). Unsurprisingly, PELP1 is largely nuclear in most studies (1). PELP1 contains ten distinct LXXLL motifs (L is leucine, x is any residue, also known as nuclear receptor box), which enable its interaction with SRs (18). Interaction of PELP1 has been documented with ER, androgen receptor (AR) (11, 19), estrogen-related receptor- (20), and retinoid X receptor (RXR) (4) and may be enhanced with ligand binding to the SR (1, 14). Each LXXLL motif within PELP1 has unique flanking sequences, which may dictate a differential affinity for each SR (21). For example, ER preferentially interacts with the fourth and fifth LXXLL motifs on PELP1 (21). RSL3 supplier In addition, PELP1 may interface with SRs through motifs other than the LXXLL motif, for example, PELP1 interacts with glucocorticoid receptor (GR) through its proline- and glutamic acid-rich regions (12). PELP1 also has several potential Src-binding sites, which account for its initial isolation inside a Src-pull down. Mechanistic studies have shown that a proline-rich region in the N terminus of PELP1 (PxxP, where P is definitely a proline, x is definitely any residue) interacts with the SH3 motif of c-Src. In addition, the SH2 motifs of PELP1 represent high-affinity connection sites for c-Src. The close proximity of nuclear receptor boxes and.