Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres

Citation
Ma. Iannone et al., Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres, CYTOMETRY, 44(4), 2001, pp. 326-337
Citations number
36
Categorie Soggetti
Medical Research Diagnosis & Treatment
Journal title
CYTOMETRY
ISSN journal
01964763 → ACNP
Volume
44
Issue
4
Year of publication
2001
Pages
326 - 337
Database
ISI
SICI code
0196-4763(20010801)44:4<326:MMIONR>2.0.ZU;2-G
Abstract
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.