Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres.
BACKGROUND
We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins.


METHODS
In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence.


RESULTS
We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen).


CONCLUSIONS
This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.
Iannone
M A
MA
Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline, Research Triangle Park, North Carolina 27709-3398, USA. mai49583@gsk.com
Consler
T G
TG
Pearce
K H
KH
Stimmel
J B
JB
Parks
D J
DJ
Gray
J G
JG
D020816:Amino Acid Motifs / Q000502:physiology; D001577:Benzophenones / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D047629:Estrogen Receptor beta; D051548:Histone Acetyltransferases; D006801:Humans; D008024:Ligands; D008863:Microspheres; D008969:Molecular Sequence Data; D056920:Nuclear Receptor Coactivator 1; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D020849:Raloxifene Hydrochloride / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000187:drug effects / Q000378:metabolism*; D013629:Tamoxifen / Q000494:pharmacology; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D001577:Benzophenones; D017362:Cyclic AMP Response Element-Binding Protein; D047629:Estrogen Receptor beta; D008024:Ligands; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D011960:Receptors, Estrogen; D014157:Transcription Factors; D013629:Tamoxifen; D014443:Tyrosine; D020849:Raloxifene Hydrochloride; D004958:Estradiol; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; C120099:GW 1929