Structure, Affinity, and Availability of Estrogen Receptor Complexes in the Cellular Environment

Abstract

An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated För-ster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor ␣-isoform (ER␣) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER␣ with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER␣s at low free SRC-RID concentrations (<2 nM) to lower affinity complexes with an ER␣ monomer at higher SRC-RID concentrations (ϳ10 nM). The methods also showed that only a subpopulation of ER␣ was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment.The dynamic processes that are fundamental to life consist of intersecting webs of interactions among biologic factors and cellular signaling pathways (1, 2). Historically, details of interaction kinetics have been measured in test tube studies that compare the level of interaction between purified factors in relationship to the amounts of those purified factors. The extent to which these quantifiers of interaction are modified when challenged by the complex environment and structure of the living cell remains an open question (3-5). For example, an ability to measure the biochemical details of estrogen action within different cellular environments may be necessary to understand tissue-specific estrogen physiology. An ability to perform cellular biochemistry also may improve our knowledge of, and treatments for, the aberrations of estrogen signaling associated with diseases such as breast or uterine cancers (6, 7). This would involve understanding how interactions of the estrogen receptors with any of a large number of cofactors (8 -11) are impacted by cell environment in different tissues of the body.Current techniques that detect interactions between factors in cells include co-immunoprecipitation, two-hybrid analysis, and Förster resonance energy transfer (FRET) 2 microscopy. FRET microscopy senses when factors co-localized in a cellular domain interact in a way that brings fluorophores attached to the factors into close enough proximity to allow energy to transfer from a Donor fluorophore to an Acceptor fluorophore (12-17). All of these methods commonly are interpreted in a simplistic binary "interaction or no interaction" fashion that does not describe quantitative differences among interactions. New technologies are needed to quantify the kinetics of interactions directly in the cell, where the rate and amounts of complexes formed may be affected by the intracellular distributions of the int...

Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy

“…The ‘3-6-1’ stable HeLa cell line, described previously [17], was maintained in DME-H21 supplemented with 5% calf serum. G418 and blasticidin were added to 700 µg/ml and 25 µg/ml, respectively, final concentrations in the media.…”

“…The HeLa cell line was previously estimated to express, on average, ~1400 CFP-AR-YFP reporter proteins in each cell [17]. That low level expression makes this an excellent cell line for establishing methods to improve FRET measurement under poor signal-to-noise conditions.…”

“…That low level expression makes this an excellent cell line for establishing methods to improve FRET measurement under poor signal-to-noise conditions. As the amounts of energy transferred from CFP to YFP, attached to the Androgen Receptor (AR), differ when different AR ligands are added to the cell culture media [17, 19], the ability to accurately measure those differences under challenging image collection conditions is the object of the current study.…”

“…Some of the variation may be due to genuine, cell-to-cell variations, which is part of the reason for conducting FRET analysis in cells. Much of the biologic variation also originates from biochemical considerations that affect FRET levels in cells that express different levels of the FP-tagged interacting factors [15–17]. By contrast, the most common non-biologic source of FRET variation originates with measurement errors inherent when quantifying a weak fluorescence signal above a high and variable background.…”

Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy

“…Hoppe and co-workers have developed an intensity based imaging methods allowing the quantification of ratio of bound and free proteins in equilibrium [48]. Subsequently, other investigators have extended this work by taking into account of the law of mass action to quantify the dissociation constant of interacting proteins [49, 50]. One limitation of these studies is that while relative dissociation constants can be determined, the absolute magnitudes cannot be measured.…”