Hynes Thomas R, Hughes Thomas E, Berlot Catherine H
Weis Center for Research, Geisinger Clinic, Danville, PA, USA.
Methods Mol Biol. 2004;237:233-46. doi: 10.1385/1-59259-430-1:233.
Heterotrimeric G proteins transmit signals from a wide range of cell surface G protein-coupled receptors (GPCRs) to mediate multiple cellular events. Within the plasma membrane, G proteins interact with GPCRs and effector proteins such as adenylyl cyclase (AC) and phospholipase C (PLC). Plasma membrane subdomains (e.g., lipid rafts and caveolae) may organize and regulate these interactions. G protein subunits have been reported to be in additional cellular regions, such as the Golgi apparatus and the cytoskeleton, and G protein alpha subunits may move within the cell during the activation cycle. Changes in the cellular localization of alpha subunits could be important for interactions with effectors that are not in the plasma membrane and/or could be a means for terminating G protein signaling. However, until recently, the topic of G protein alpha subunit localization under basal and activated conditions has been controversial, partly because of spatial and temporal limitations inherent to procedures like cell fractionation and immunohistochemistry. Green fluorescent protein (GFP)-tagging is a useful way to enable real-time visualization of proteins in living cells. This chapter describes how to produce and visualize functional GFP-tagged alpha subunits and to investigate whether activation affects their subcellular localization.
