Fluorescence resonance energy transfer to study receptor dimerization in living cells.
The versatility, sensitivity, and feasibility of fluorescence methods are very attractive to study protein-protein interaction at low levels of protein expression. However, one of the most severe limits in protein chemistry has been the difficulty of introducing site-specific fluorescent labels. The development of genetically encoded fluorescent probes, that is, green fluorescent protein (GFP) and its variants therefore opened up a broad field of novel applications. To characterize protein-protein interactions and determine detailed spatio-temporal dynamics of partners that are molecularly well characterized, fluorescence energy transfer methods are excellent nondestructive tools in living cells. Cellular responses to external factors are extensively based on direct molecular interaction and especially G-protein-coupled receptors (GPCRs) have been shown to interact with an unexpected level of complexity. Classical models of signal transduction describe GPCRs as monomeric proteins, while recent studies using fluorescence resonance energy transfer (FRET) and other methods show that GPCRs can also function as homo- or heterodimers. Theoretical background information on FRET technology and its diverse applications are summarized here. A detailed description of a spectroscopic method for FRET studies in the field of GPCR interaction is presented to facilitate and propagate studies to increase our understanding of protein-protein interactions involving GPCRs.
Bader
Jürgen E
JE
Institute of Biochemistry, University of Leipzig, Germany.
Beck-Sickinger
Annette G
AG
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D017476:Receptors, Neuropeptide Y; D049452:Green Fluorescent Proteins