Abstract
Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) for α subunits of the G i andor G q class of heterotrimeric G proteins. RGS GAP activity is inhibited by phosphatidic acid (PA), lysophosphatidic acid (LPA), and phosphatidylinositol 3,4,5-trisphosphate (PIP 3) but not by other phospholipids, phosphoinositides, or diacylglycerol. Both PA and PIP 3 can inhibit RGS4 GAP activity and their inhibition is additive, suggesting that PA and PIP 3 interact with different domains of RGS4. The N terminus of RGS4 (1-57 amino acids) is required for PA binding and inhibition. Mutation at Lys20, far from the RGS domain of RGS4, decreases PA-mediated inhibition of RGS4 by more than 85%. Amino acid substitutions in helix 5 within the RGS domain of RGS4, opposite to the RGSGα protein contact face, reduce binding affinity and inhibition by PIP 3. Calmodulin binds all RGS proteins tested in a Ca 2+-dependent manner at two sites, one in the N-terminal 33 amino acids and another in the RGS domain. Ca 2+calmodulin does not directly affect GAP activity of RGS4 but reverses PA and PIP 3-mediated inhibition. In summary, these results demonstrate that phospholipids such as PA and PIP 3 act as allosteric inhibitors of RGS proteins, and Ca 2+calmodulin competition with PA and PIP 3 may provide an intracellular mechanism for feedback regulation of Ca 2+ signaling evoked by G-protein-coupled agonists.