Chapter 19 Measuring the Proximity of T‐Lymphocyte CXCR4 and TCR by Fluorescence Resonance Energy Transfer (FRET)

Multiprotein complexes play an important role in nearly all cell functions; therefore, the characterization of protein-protein interactions in living cells constitutes an important step in the analysis of cellular signaling pathways. Using fluorescence resonance energy transfer (FRET) as a "molecular ruler" is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. Here, we describe two methods that use FRET to detect a physical interaction between the T-cell antigen receptor (TCR) and the CXCR4 chemokine receptor in living T lymphocytes. These FRET approaches use two different sets of chromophores. We discuss the design strategies, control experiments, and pitfalls involved in using these FRET approaches. Although there is no perfect pair of chromophores for FRET, the two FRET methods described here provide complementary and reliable insight into the molecular interactions between these receptor molecules.