PMID- 12964159
OWN - NLM
STAT- MEDLINE
DCOM- 20040507
LR  - 20061115
IS  - 1439-4227 (Print)
IS  - 1439-4227 (Linking)
VI  - 4
IP  - 9
DP  - 2003 Sep 5
TI  - Fluorescent cellular sensors of steroid receptor ligands.
PG  - 848-55
AB  - Steroid hormone receptors comprise a major class of therapeutic drug targets that 
      control gene expression by binding steroid hormone ligands. These small 
      molecule-protein interactions are typically characterized in living cells by 
      quantification of ligand-mediated reporter gene expression. As an alternative, 
      non-transcriptional approach, we constructed fluorescent cellular sensors by 
      expressing yellow fluorescent protein (YFP) fused to the ligand binding domains 
      (LBDs) of estrogen receptor-alpha (ERalpha), estrogen receptor-beta (ERbeta), 
      androgen receptor (AR), and the glucocorticoid receptor (GR). These proteins were 
      tethered through a short two amino acid linker and expressed in S. cerevisiae 
      yeast. Recombinant yeast treated with cognate steroid receptor ligands exhibited 
      dose-dependent fluorescence enhancements that were correlated with known relative 
      receptor binding affinity values. These effects generally paralleled 
      ligand-mediated receptor dimerization quantified with analogous yeast two-hybrid 
      transcriptional assays, suggesting that the majority of the observed fluorescence 
      enhancements were conferred by conformational changes coupled with receptor 
      dimerization, such as ligand-mediated stabilization of protein folding. 
      Remarkably, certain interactions such as the binding of cortisol, progesterone, 
      and dexamethasone to the GR were undetectable with yeast two-hybrid assays. 
      However, these interactions were detected with the fluorescent cellular sensors, 
      indicating the sensitivity of this system to subtle ligand-induced conformational 
      effects. These sensors provide a novel, non-transcriptional, and high-throughput 
      method to identify and analyze ligands of nuclear hormone receptors.
FAU - Muddana, Smita S
AU  - Muddana SS
AD  - Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, 
      University Park, PA 16802, USA.
FAU - Peterson, Blake R
AU  - Peterson BR
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Germany
TA  - Chembiochem
JT  - Chembiochem : a European journal of chemical biology
JID - 100937360
RN  - 0 (Ligands)
RN  - 0 (Receptors, Steroid)
RN  - 0 (Recombinant Fusion Proteins)
SB  - IM
MH  - Animals
MH  - Binding, Competitive
MH  - *Biosensing Techniques
MH  - Flow Cytometry
MH  - Fluorescence
MH  - Humans
MH  - Ligands
MH  - Rats
MH  - Receptors, Steroid/genetics/*metabolism
MH  - Recombinant Fusion Proteins/genetics/metabolism
MH  - Saccharomyces cerevisiae/genetics
MH  - Substrate Specificity
MH  - Two-Hybrid System Techniques
EDAT- 2003/09/10 05:00
MHDA- 2004/05/08 05:00
CRDT- 2003/09/10 05:00
PHST- 2003/09/10 05:00 [pubmed]
PHST- 2004/05/08 05:00 [medline]
PHST- 2003/09/10 05:00 [entrez]
AID - 10.1002/cbic.200300606 [doi]
PST - ppublish
SO  - Chembiochem. 2003 Sep 5;4(9):848-55. doi: 10.1002/cbic.200300606.