Homo- and Hetero-FRET Imaging of Membrane Proteins in Live Cells

Abstract

The structure-function-activity relationships of transmembrane receptors are often mediated not only by ligand-induced signaling but also homo- and hetero-philic binding interactions. Understanding the molecular basis for these interactions is therefore critical for elucidating receptor function. We are using live cell total internal reflection fluorescence microscopy (TIRFM) to examine the distribution, association, and ligand accessibility of (1) carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1), and (2) fibroblast growth factor receptor 1 (FGFR1), which is thought to associate with FGF21 co-receptor Klotho-beta (KLB). By combining TIRFM with homo- and hetero- Forster Resonance Energy Transfer (homo-FRET and hetero-FRET, respectively), we are able to track the real-time response of CEACAM1 oligomerization and dynamics to soluble factors (such as CEACAM-specific antibody, ionomycin, and soluble CEACAM1), as well as KLB's and FGFR1's responses to FGF21. Obtaining a better understanding of the molecular dynamics of CEACAM1, KLB, and FGFR1 as well as their spatial and oligomeric distributions is important for elucidating their roles in downstream signaling pathways.