Determination of the affinities between heterotrimeric G protein subunits and their phospholipase C-beta effectors.

Phosphatidylinositide-specific phospholipase C-betas play a key role in Ca2+ signaling and are specifically activated by the alphaq family of heterotrimeric G proteins and as well as betagamma subunits. We have determined the affinity between Gbetagamma subunits and GTPgammaS and GDP-liganded Galphaq subunits on membrane surfaces, and their respective affinities to PLC-beta1, -beta2 and -beta3 effectors by fluorescence spectroscopy. We find that activation of Galphaq by GTPgammaS decreases its affinity for Gbetagamma subunits at least 36-fold compared to the GDP-liganded form, but increases its affinity for PLC-betas at least 40-200-fold depending on the PLC-beta isoform. The affinity of Galphaq(GTPgammaS) is similar for PLC-beta1 and -beta3 and 10-fold stronger for PLC-beta2, which corresponds to the reported relationship between the concentration of Galphaq(GTPgammaS) and PLC-beta activation on lipid bilayers. We find that a large portion of the PLC-beta-Galphaq association energy lies within the 400 residue C-terminal region of PLC-beta1 since truncating this region reduces its Galphaq affinity. In contrast, the isolated N-terminal region does not interact with Galphaq. Gbetagamma subunits interact with all three PLC-beta isotypes, but only showed strong binding to PLC-beta2, and activation of the three PLC-betas by Gbetagamma subunits parallels this behavior. We also tested the possibility that both Galphaq and Gbetagamma can simultaneously bind PLC-beta2. Our data argue against simultaneous binding and show that Galphaq and Gbetagamma independently regulate this effector.