Detection of phospholipase C-β{sub}2 activation by G-protein subunits

摘要

Many neurotransmitters and hormones convey their signals into cells via transmembrane receptors that activate heterotrimeric G proteins which in turn activate phospholipase C-β(PLC-β).Activation of PLC-β by the α{sub}q and β{sub}γ subunits of G proteins results in activation of protein kinase C and release of Ca{sup}(2+) from intracellular stores, which in turn results in a multitude cellular changes. We have recently found that activation of PLC-β by G proteins occurs by lateral association on the membrane surface. Here, we have measured the affinity of the membrane-bound species by fluorescence energy transfer and have conducted time-resolved studies to assess the lifetime of the PLC-G protein complexes in order to understand PLC signaling inside the cell. To better interpret these results, we outline methods to convert the two-dimensional dissociation constant measured for the membrane-bound proteins to a three dimensional one. We also detail calculations to determine the concentrations at which non-specific protein-protein interactions occurs due to membrane crowding. To differentiate between the physical association of PLC-G complexes and activation, we have developed a real-time fluorescence-based PLC-β activity to determine the length of time that PLC-β remains active after G protein dissociation. A model for activation will be presented.