Background: We describe a novel microsphere-based system to identify and ch
aracterize 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 peptid
es. The coactivator peptide-coupled microsphere populations are combined an
d incubated with a nuclear receptor that has been coupled to a green fluoro
chrome. Flow cytometric analysis of the microspheres simultaneously decodes
each population and detects the binding of receptor to respective coactiva
tor peptides by the acquisition of green fluorescence.
Results: We have used this system to determine the binding affinities of hu
man estrogen receptor beta ligand binding domain (ERbeta IBD) and human per
oxisome proliferator activated receptor gamma ligand binding domain (PPAR g
amma LBD) to a set of 34 coactivator peptides. Binding of ER beta LBD to a
coactivator peptide sequence containing the second LXXLL motif of steroid r
eceptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and satur
able. Analysis of receptor binding to a multiplexed set of coactivator pept
ides shows PPAR gamma LBD binds with high affinity to cAMP response element
binding protein (CBP) peptides and to the related P300 peptide while ER be
ta LBD exibits little binding to these peptides. Using the microsphere-base
d assay we demonstrate that ER beta LBD and PPAR gamma LBD binding affiniti
es for the coactivator peptides are increased in the presence of agonist (e
stradiol or GW1929, respectively) and that ER beta LBD binding is decreased
in the presence of antagonist (raloxifene or tamoxifen).
Conclusions: This unique microsphere-based system is a sensitive and effici
ent method to simultaneously evaluate many receptor-coactivator interaction
s in a single assay volume. In addition, the system offers a powerful appro
ach to study small molecule modulation of nuclear receptor binding. Cytomet
ry 44:326-337, 2001. (C) 2001 Wiley-Liss, Inc.