Heterotrimeric G proteins are activated by membrane receptors that respond to the presence of extracellular hormones, neurotransmitters and growth factors. G proteins can then activate one or more effectors molecules by an unknown mechanism which then determines the pathway the signal will travel. Cells contain many receptors, effectors and G proteins that can interact. The affinity and duration of a particular G-effector complex is expected to underlie the particular signal pathway, but these parameters have never been measured for any G protein and effector. Our long term goal is to understand the mechanism through which specific G protein subtypes activate specific effectors. In this proposal two distinct mechanisms of effector activation will be tested. Also, the affinity and lifetime of the complexes, and the duration of the activated state of the effector will be determined. We will focus on the effectors, phospholipase beta1 (PLCbeta1), which is primarily activated by the alpha subunits of the G-q class of proteins, and PLCbeta2, which is primarily activated by the beta-gamma subunits. PLCbeta plays a pivotal role in signal transduction and disruptions in this pathway is associated with disease states of the cell. The major experimental technique we will use to achieve these goals is fluorescence spectroscopy. Fluorescence methods will allow us to view protein associations and conformational changes in real time, and will allow us to assess multiple equilibria. Moreover, fluorescence will allow us to quantitate the energies and time scales of these interactions. After measuring the affinities and lifetimes of complexes formed between G-alpha, G-beta-gamma and PLC-beta1 and PLCbeta2 using steady state and stop-flow methods, we will determine whether PLCbeta activation occurs by recruitment of the protein to the membrane surface by G proteins, or whether activation is due to association of the membrane-bound species. We will then determine whether PLCbeta activation occurs through a conformational change, and whether this activated conformation is sustained upon dissociation of the G protein subunit. Secondary factors that may serve to regulate PLCbeta activation will then be investigated such as the nature of the membrane surface and simultaneous association of both G protein subunits to the different PLC isoforms.
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