Kinetics of G-protein-coupled receptor signals in intact cells

Abstract

G-protein-coupled receptors (GPCRs) are the largest group of cell surface receptors. They are stimulated by a variety of stimuli and signal to different classes of effectors, including several types of ion channels and second messenger-generating enzymes. Recent technical advances, most importantly in the optical recording with energy transfer techniques--fluorescence and bioluminescence resonance energy transfer, FRET and BRET--, have permitted a detailed kinetic analysis of the individual steps of the signalling chain, ranging from ligand binding to the production of second messengers in intact cells. The transfer of information, which is initiated by ligand binding, triggers a signalling cascade that displays various rate-controlling steps at different levels. This review summarizes recent findings illustrating the speed and the complexity of this signalling system.

Figure 1

Example of a G-protein-coupled receptor modified for fluorescence resonance energy transfer (FRET) analysis of receptor activation (conformational change). A cyan fluorescent protein (CFP) is placed in the third intracellular loop and a yellow fluorescent protein (YFP) at the C terminus. FRET occurs upon excitation of CFP (with light at 436 nm, causing emission at 480 nm), which allows energy transfer to YFP (provided that the latter is close enough, <10 nm distance), which results in emission at 535 nm. The ratio of 535 and 480 nm emissions is an indicator of FRET and is strongly influenced by the distance between CFP and YFP, serving as a kind of ‘molecular ruler'. It should be noted that emission of CFP and/or YFP can also be affected by other factors, such as pH or fluorescence quenching, which necessitates appropriate controls. In bioluminescence resonance energy transfer (BRET), the donor CFP is replaced by the light-emitting enzyme luciferase. In a variant of FRET, the acceptor YFP is replaced by the small dye FlAsH, which binds to tetracysteine-containing motifs that can be engineered into the protein sequence.