Precoupling of G Proteins with GPCRs Visualized by Two-Photon Polarization Microscopy

Abstract

G protein-coupled receptors (GPCRs) and G proteins are key players of cellular signal transduction. They transduce signals from a multitude of extracellular stimuli into the cells. Despite a number of studies, many aspects of molecular mechanisms and spatiotemporal dynamics of G protein interactions with GPCRs remain unclear. In particular, it is uncertain whether G proteins can form complexes with inactive GPCRs, and what is the stability and functional significance of such complexes.In order to address these issues, we have now investigated the interactions between G proteins of the Gi family and various GPCRs using the technique of two-photon polarization microscopy (2PPM). 2PPM, developed in our laboratory, allows sensitive monitoring of protein conformational changes and protein-protein interactions, in living cells, in real time, using a single fluorescent protein tag. Our results demonstrate that 2PPM indeed allows observing interactions between Gi proteins and GPCRs using a single fluorescent protein tag. Furthermore, our results reveal distinct types of interaction between Gi proteins and the studied GPCRs. Finally, our results allow us to distinguish between interactions of non-stimulated GPCRs with G proteins which lead to G protein activation (precoupling), and formation of complexes between non-stimulated GPCRs and G proteins without further G protein activation (preassembly). We can, therefore, make conclusions about the mechanisms of GPCR-G protein signal transduction.