Review - Using green fluorescent protein to study intracellular signalling

Subcellular compartmentalisation of signalling molecules is an important ph enomenon riot only in defining how a signalling pathway is activated but al so in influencing the desired physiological output of that pathway (e.g. ce ll growth or differentiation, regulation of metabolism, cytoskeletal change s etc.). Biochemical analyses of protein and lipid compartmentalisation by, for example, subcellular fractionation presents many technical difficultie s. However, this aspect of cell signalling research has seen a major revolu tion thanks to the cloning and availability of a variety of mutant green fl uorescent protein derivatives with distinct molecular properties. Mutants w ith increased brightness, altered excitation and emission maxima, altered s tability 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 o f the recent developments in the use of green fluorescent proteins for stud ying 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 fluoresc ence resonance energy transfer.