Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET 2 )

Abstract

Bioluminescent resonance energy transfer (BRET²) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET² is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP²)). The BRET² assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET² signal. BRET² is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET² signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET² in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET². This article describes in detail the BRET² methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

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