Abstract
Estrogen receptor α (ER-α) is a nuclear hormone receptor that controls selected genes, thereby regulating proliferation and differentiation of target tissues, such as breast. Gene expression controlled by ER-α is modulated by Ca2+ via calmodulin (CaM). Here we present the NMR structure of Ca2+-CaM bound to two molecules of ER-α (residues 287-305). The two lobes of CaM bind to the same site on two separate ER-α molecules (residues 292, 296, 299, 302, and 303), which explains why CaM binds two molecules of ER-α in a 1:2 complex and stabilizes ER-α dimerization. Exposed glutamate residues in CaM (Glu-11, Glu-14, Glu-84, and Glu-87) form salt bridges with key lysine residues in ER-α (Lys-299, Lys-302, and Lys-303), which is likely to prevent ubiquitination at these sites and inhibit degradation of ER-α. Transfection of cells with full-length CaM slightly increased the ability of estrogen to enhance transcriptional activation by ER-α of endogenous estrogen-responsive genes. By contrast, expression of either the N- or C-lobe of CaM abrogated estrogen-stimulated transcription of the estrogen responsive genes pS2 and progesterone receptor. These data suggest that CaM-induced dimerization of ER-α is required for estrogen-stimulated transcriptional activation by the receptor. In light of the critical role of ER-α in breast carcinoma, our data suggest that small molecules that selectively disrupt the interaction of ER-α with CaM may be useful in the therapy of breast carcinoma.
Highlights
Estrogen receptor ␣ (ER-␣) is a nuclear hormone receptor that controls selected genes, thereby regulating proliferation and differentiation of target tissues, such as breast
Western blotting of cell lysates verified the expression of equivalent levels of Previously, we solved the NMR structures of individual CaM lobes (CaM-N and CaM-C) each of which binds to the same site on ER-␣ [15]
On the basis of this earlier structural analysis, we proposed that each lobe of CaM binds to a separate ER-␣ molecule
Summary
NMR Structure of Full-length CaM Bound to Two Molecules of ER-␣—Previously we solved NMR structures of individual CaM lobes (CaM-N and CaM-C) each bound to a peptide fragment of ER-␣ (residues 287–305, called ER[287–305]) [15]. The 1H-15N HSQC spectrum of 15N-labeled CaM bound to two molecules of ER[287–305] (Fig. 1A) looks similar to the NMR spectra of CaM-N (cyan in Fig. 1A) and CaM-C (magenta in Fig. 1A) each bound to one molecule of ER[287–305] This spectral similarity demonstrates that in the full-length protein both CaM lobes are independently folded and each lobe binds independently to a separate ER-␣ molecule. GFP-tagged full-length CaM bound to endogenous ER-␣ in MCF-7 cell lysates (Fig. 3A) Probing the blots for GFP showed similar amounts of each CaM construct (Fig. 3A, lower panel) These data reveal that the N- and C-halves of CaM independently bind ER-␣ in cell lysates. We tagged CaM-F, CaM-N, and CaM-C with GST, expressed the proteins in Escherichia coli (Fig. 3B, lower panel) and incubated each with equal amounts of MCF-7 cell lysate. Hydrophobic side chain atoms in ER-␣ (Trp-292) form detailed contacts with each lobe of CaM, and basic side chains in ER-␣ (Lys-299 and Lys-303) form salt bridges with Glu-14 (N-lobe) and Glu-84 (C-lobe) of CaM
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