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Estrogens, acting via estrogen receptor (ER) play key roles in growth, differentiation and gene regulation in the reproductive, central nervous and skeletal systems. ER-mediated gene transcription contributes to the development and spread of breast, uterine, and liver cancer. Steroid receptor coactivator-1a (SRC1a) belongs to the P160 family of coactivators, which is the best known of the many coactivators implicated in ER-mediated transactivation. Binding of full-length P160 coactivators to steroid receptors has been difficult to investigate in vitro. This chapter details how to investigate the interaction of SRC1a with ER using the fluorescence anisotropy/polarization microplate assay (FAMA).
Estrogens regulate the normal growth and differentiation of reproductive tissues, bone and nervous system and play an important role in human pathologies such as osteoporosis and breast, uterine, ovarian liver and lung cancer (1–2). Estrogens, such as 17β-estradiol (E2), execute their genomic actions mainly through estrogen receptor (ER) (3). There are two estrogen receptor isoforms, ERα and ERβ with substantial differences in the N-terminal domain and ligand binding domain (LBD) (4–5). In the classical mechanism of ER action, the ER dimerizes when bound to E2, and the E2-ER complex regulates gene expression by direct binding to estrogen response elements (EREs), or closely related sequences, (6–8). The consensus ERE (cERE) is a perfect palindrome, but most functional EREs are not perfect palindromes (9). For example, the downstream regulatory region of the proteinase inhibitor 9 (PI-9) gene called the estrogen responsive unit (ERU) consists of both an imperfect ERE palindrome and a direct repeat (10). Once bound to an ERE, the ligand binding domain of ER assumes a conformation that enables the recruitment of coactivators (11). Bound coactivators help assemble a multi-protein complex that facilitates both chromatin remodeling and formation of an active transcription complex.
The steroid receptor coactivator (SRC) or p160 family of coactivators, including SRC1/NCoA-1, SRC2/TIF2/GRIP1/NCoA-2 and SRC3/pCIP/ACTR/AIB1 represents a major class of coactivators that play key roles in ER-mediated transcription (12). The p160 coactivators interact with the hydrophobic binding cleft in the ER LBD mainly via highly conserved α-helical Leu-x-x-Leu-Leu (LxxLL) motifs, also called nuclear receptor (NR) boxes (13). All of the SRCs contain a central nuclear receptor-interacting domain (NRID) made up of three NR boxes separated by ~55 amino acids. SRC1a is an alternatively spliced form of SRC1 and contains a fourth NR box at its C-terminus (14).
We developed the fluorescence anisotropy/polarization microplate assay (FAMA) to analyze the interactions of steroid receptors with their DNA recognition sites (15) and with coactivators, such as full length SRC1a (16). In addition FAMA is a facile technology for high throughput screening for small molecule inhibitors of receptor interactions (17–19).
In this assay, ER is pre-bound to a fluorescein-labeled ERE (flERE) in ultra low-volume microplates (16). When polarized light excites the flERE, most of the emitted light is depolarized because of rapid rotational diffusion of the flERE that results in its position being largely randomized at the time of emission. Binding of the ER protein, or of the even larger full-length SRC1a-ER complex, to the flERE slows rotation of the flERE, increasing the possibility that the complex is in the same plane at emission and excitation. These changes can be observed as an increase in fluorescence polarization, or the closely-related parameter, fluorescence anisotropy (detailed in Fig. 1). The coactivator binding assay described here is the most sophisticated application of the FAMA.
This experiments tests the ability of SRC1a to bridge, or link, two ER dimers bound at a complex DNA binding site. Linking the two DNA-bound ER dimers will increase the stability of the ERDNA complex.
1Avoid generating bubbles when mixing the samples in the microplate. Bubbles can alter the readings.
2Many fluorescence polarization/anisotropy plate readers lack the sensitivity required to accurately measure polarization in small 10–20 μl volumes. If the plate-reader does not directly show anisotropy as a read-out, the anisotropy can be calculated from the raw data using the formula: FA= (I//−I)/ (I//+2I), where I// and I are the emission light intensity (I) in the parallel (//) and perpendicular () directions to the excitation light, respectively. Although outcomes were independent of the instrument, gain settings vary widely between instruments; therefore the magnitude of the anisotropy change can vary significantly in experiments performed using different plate readers.
3The activities of different batches of purified protein may vary. It is best to determine the actual concentration needed by generating a binding curve.
4The time lag between SRC1a addition and the reading of the first data point is likely to be 5–15 seconds, and depends on the speed with which the microplate reader can move a microplate into place and begin reading it. A timer can be used to count. The time intervals between each reading may be limited by the plate-reader and can be more than 2 seconds. In that case just choose the fastest mode available.