Determination of the Affinities between Heterotrimeric G Protein Subunits and Their Phospholipase C-β Effectors

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Abstract
Phosphatidylinositide-specific phospholipase C-βs play a key role in Ca2+ signaling and are specifically activated by the αq family of heterotrimeric G proteins and as well as βγ subunits. We have determined the affinity between Gβγ subunits and GTPγS and GDP-liganded Gαq subunits on membrane surfaces, and their respective affinities to PLC-β1, -β2 and -β3 effectors by fluorescence spectroscopy. We find that activation of Gαq by GTPγS decreases its affinity for Gβγ subunits at least 36-fold compared to the GDP-liganded form, but increases its affinity for PLC-βs at least 40−200-fold depending on the PLC-β isoform. The affinity of Gαq(GTPγS) is similar for PLC-β1 and -β3 and 10-fold stronger for PLC-β2, which corresponds to the reported relationship between the concentration of Gαq(GTPγS) and PLC-β activation on lipid bilayers. We find that a large portion of the PLC-β-Gαq association energy lies within the 400 residue C-terminal region of PLC-β1 since truncating this region reduces its Gαq affinity. In contrast, the isolated N-terminal region does not interact with Gαq. Gβγ subunits interact with all three PLC-β isotypes, but only showed strong binding to PLC-β2, and activation of the three PLC-βs by Gβγ subunits parallels this behavior. We also tested the possibility that both Gαq and Gβγ can simultaneously bind PLC-β2. Our data argue against simultaneous binding and show that Gαq and Gβγ independently regulate this effector.