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

Phosphatidylinositide-specific phospholipase C-beta s play a key role in Ca2+ signaling and are specifically activated by the cc, family of heterotrimeric G proteins and as well as beta gamma subunits. We have determined the affinity between G beta gamma subunits and GTP gamma S and CDP-liganded G alpha(q) subunits on membrane surfaces, and their respective affinities to PLC-beta(1), -beta(2) and -beta(3) effecters by fluorescence spectroscopy. We find that activation of G alpha(q) by GTP gamma S decreases its affinity for G beta gamma subunits at least 36-fold compared to the GDP-liganded form, but increases its affinity for PLC-beta s at least 40-200-fold depending on the PLC-beta isoform. The affinity of G alpha(q)(GTP gamma S) is similar for PLC-beta(1) and -beta(3) and 10-fold stronger for PLC beta(2), which corresponds to the reported relationship between the concentration of G alpha(q)(GTP gamma S) and PLC-beta activation on lipid bilayers. We find that a large portion of the PLC-beta-G alpha(q), association energy lies within the 400 residue C-terminal region of PLC-beta(1) since truncating this region reduces its G alpha(q) affinity. In contrast, the isolated N-terminal region does not interact with G alpha(q). G beta gamma subunits interact with all three PLC-beta isotypes, but only showed strong binding to PLC-beta(2), and activation of the three PLC-beta s by G beta gamma subunits parallels this behavior. We also tested the possibility that both G alpha(q) and G beta gamma can simultaneously bind PLC-beta(2). Our data argue against simultaneous binding and show that G alpha(q) and G beta gamma independently regulate this effector.