Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta - PubMed (original) (raw)

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta

L A Paige et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs alpha and beta and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER alpha and ER beta ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

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Figures

Figure 1

Figure 1

(A) Sequences of LXXLL motif containing peptides that were affinity selected on ER α in the presence of estradiol. (B) Sequences of LXXLL motifs found in the nuclear receptor coactivating proteins human SRC1a (steroid receptor coactivator 1a), mouse cAMP-responsive element binding protein (CREB)-binding protein (CBP), and human RIP140.

Figure 2

Figure 2

Phage ELISA. A biotinylated vitellogenin ERE was immobilized on 96-well plates precoated with streptavidin. The ER was then immobilized on the ERE and incubated for 5 min in the presence of modulator before the addition of phage. Assays were conducted as described in Materials and Methods. HRP, horseradish peroxidase.

Figure 3

Figure 3

Analysis of the binding specificity of the conformational probes was conducted by phage ELISA as described in Materials and Methods. Estradiol and 4-OH tamoxifen concentrations were 1 μM. The probes α/β I-α/β V are shown only for ER α. The binding patterns of these probes on ER β were similar. Sequences of the probes are as follows: α/β I, SSNHQSSRLIELLSR; α/β II, SAPRATISHYLMGG; α/β III, SSWDMHQFFWEGVSR; α/β IV, SRLPPSVFSMCGSEVCLSR; α/β V, SSPGSREWFKDMLSR; α I, SSEYCFYWDSAHCSR; α II, SSLTSRDFGSWYASR; α III, SRTWESPLGTWEWSR; β I, SREWEDGFGGRWLSR; β II, SSLDLSQFPMTASFLRESR; β III, SSEACVGRWMLCEQLGVSR.

Figure 4

Figure 4

Fingerprint analysis of ER modulators on (A) ER α and (B) ER β. Immobilized ER was incubated with estradiol (1 μM), estriol (1 μM), Premarin (10 μM), 4-OH tamoxifen (1 μM), nafoxidine (10 μM), clomiphene (10 μM), raloxifene (1 μM), ICI 182,780 (1 μM), 16α-OH estrone (10 μM), DES (1 μM), or progesterone (1 μM). Phage ELISAs were conducted as described in Materials and Methods.

Figure 5

Figure 5

Comparison of the binding of the peptide probes to ER α or ER β in the presence of modulators using TRF. Assays were conducted as described in Materials and Methods. (○), buffer, aporeceptor; (□) 17β estradiol;(▵) estriol; (▴) DES; (▿) 4-OH tamoxifen; (▾) raloxifene; (⋄)nafoxidine; (♦) clomiphene; (★) ICI 182,780. RFU, relative fluorescence units.

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