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 IBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPAR gamma 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 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 PPAR gamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ER beta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ER beta LBD and PPAR gamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol 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 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. Cytometry 44:326-337, 2001. (C) 2001 Wiley-Liss, Inc.