Real-time analysis of molecular interaction of retinoid receptors and receptor-interacting protein 140 (RIP140).

Abstract

Receptor interacting protein 140 (RIP140) is a coregulator for a large number of transcription factors. RIP140 interacts with retinoic acid receptor (RAR) and retinoid X receptor (RXR) with or without ligands. The C-terminal domain of RIP140 (RIP-C') contains a novel sequence (1063-1076, LTKTNPILYYMLQK) and has been shown to interact with RAR and RXR ligand dependently in two-hybrid interaction and pull-down assays. To examine the kinetic characteristics of molecular interaction of RIP-C' with RAR and RXR, a surface plasmon resonance technology (BIAcore) was applied for real-time analyses of this molecular interaction with highly purified proteins. A modified pull-down assay using purified proteins was also conducted to obtain supporting data. The effect of retinoid ligands on this type of interaction was addressed. By using receptor mutants, it was demonstrated that the activation function-2 domain and the ability to form dimers of the receptors are required for an efficient interaction of receptor with RIP140. Finally, with a mutagenesis approach, we determined the effects of specific point mutations on the kinetics of RIP-C' interaction with RAR/RXR.