Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase Cβ₁

Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three members of the G protein-phospholipase Cβ (PLC-β) signaling cascade which may allow for rapid deactivation of intracellular Ca²⁺ release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-β, G-βγ, and both deactivated (GDP-bound) and activated (GTPγS-bound) Gα_q. We find that RGS4 not only binds to activated Gα_q, as predicted, but also to Gβγ and PLCβ₁. These interactions occur through protein-protein contacts since the intrinsic membrane affinity of RGS4 was found to be very weak in the absence of the protein partner PLCβ₁ or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Gα_q, Gβγ and phospholipase Cβ₁ will form, but only at relatively high protein concentrations. We propose that these interactions allow RGS4 to remain anchored to the signaling complex even in the quiescent state and allow rapid transfer to activated Gα_q to shut down the signal. Comparison of the relative affinities between these interacting proteins will ultimately allow us to determine whether certain complexes can form and where signals will be directed.