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PMID: 11390668 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells.

Molecular and cellular biology ·Vol. 21 ·No. 13 ·2001-07-00 ·Pages 4404-12

Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M, O'Malley BW, Smith CL, Belmont AS, Mancini MA

Abstract

Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor alpha (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-tagged lac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integrated lac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP-SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP-SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP-SRC-1, while antagonist additions diminish YFP-SRC-1-CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER-SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP-SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

MeSH Terms
Amino Acid Motifs Animals Binding Sites Cell Line Estrogen Receptor alpha Estrogens/pharmacology Genes, Reporter Histone Acetyltransferases Lac Operon/genetics Ligands Microscopy, Fluorescence Nuclear Receptor Coactivator 1 Protein Binding/drug effects Receptors, Estrogen/genetics,metabolism Recombinant Fusion Proteins/genetics,metabolism Regulatory Sequences, Nucleic Acid/genetics Trans-Activators/genetics,metabolism Transcription Factors/genetics,metabolism Transfection
Chemicals
Estrogen Receptor alpha Estrogens Ligands Receptors, Estrogen Recombinant Fusion Proteins Trans-Activators Transcription Factors Histone Acetyltransferases Nuclear Receptor Coactivator 1
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Stenoien D L
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Nye A C
Mancini M G
Patel K
Dutertre M
O'Malley B W
Smith C L
Belmont A S
Mancini M A
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.21.13.4404-4412.2001
Published
2001-07-00
Pages
4404-12
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC87100
Subset
IM
Grants
NIGMS NIH HHS · T32-GM07283 · United States
NIDDK NIH HHS · R01-DK55622 · United States
NIDDK NIH HHS · R01 DK53002 · United States
NIDDK NIH HHS · R01 DK055622 · United States
NIGMS NIH HHS · R01 GM042516 · United States
NIDDK NIH HHS · R01 DK053002 · United States
NIGMS NIH HHS · R01-GM58460 · United States
NIDDK NIH HHS · F32 DK009787 · United States
NIGMS NIH HHS · T32 GM007283 · United States
NIGMS NIH HHS · R01 GM058460 · United States
NIDDK NIH HHS · 1F32DK09787 · United States
NIGMS NIH HHS · R01-GM42516 · United States
External Links
PubMed source DOI: 10.1128/MCB.21.13.4404-4412.2001 PMC Full Text View journal details
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