Structure, affinity, and availability of estrogen receptor complexes in the cellular environment.
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 Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor alpha-isoform (ER alpha) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER alpha with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER alphas at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ER alpha monomer at higher SRC-RID concentrations (approximately 10 nm). The methods also showed that only a subpopulation of ER alpha 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.
Kofoed
Eric M
EM
Diabetes Center, University of California, San Francisco, California 94143-0540, USA.
Guerbadot
Martin
M
Schaufele
Fred
F
D001671:Biochemistry / Q000379:methods*; D002138:Calibration; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000378:metabolism / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008956:Models, Chemical; D056920:Nuclear Receptor Coactivator 1 / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014162:Transfection
D047628:Estrogen Receptor alpha; D004967:Estrogens; D004958:Estradiol; D056920:Nuclear Receptor Coactivator 1