Using green fluorescent protein to study intracellular signalling

Subcellular compartmentalisation of signalling molecules is an important phenomenon not only in defining how a signalling pathway is activated but also in influencing the desired physiological output of that pathway (e.g. cell growth or differentiation, regulation of metabolism, cytoskeletal changes etc.). Biochemical analyses of protein and lipid compartmentalisation by, for example, subcellular fractionation presents many technical difficulties. However, this aspect of cell signalling research has seen a major revolution thanks to the cloning and availability of a variety of mutant green fluorescent protein derivatives with distinct molecular properties. Mutants with increased brightness, altered excitation and emission maxima, altered stability and differential sensitivity to pH, are now in widespread use for following the trafficking and function of proteins in living cells and for monitoring the intracellular environment. In this review we focus on some of the recent developments in the use of green fluorescent proteins for studying intracellular signalling pathways often with special reference to the actions of insulin. We also discuss the future utility of these proteins to analyse protein--protein interactions in signalling pathways using fluorescence resonance energy transfer.