Phosphatidylinositol 3,4,5-trisphosphate and Ca²⁺/calmodulin competitively bind to the regulators of G-protein-signalling (RGS) domain of RGS4 and reciprocally regulate its action

RGS (regulators of G-protein signalling) are a diverse group of proteins, which accelerate intrinsic GTP hydrolysis on heterotrimeric G-protein α subunits. They are involved in the control of a physiological behaviour known as ‘relaxation’ of G-protein-gated K⁺ channels in cardiac myocytes. The GTPase-accelerating activity of cardiac RGS proteins, such as RGS4, is inhibited by PtdIns(3,4,5)P₃ (phosphatidylinositol 3,4,5-trisphosphate) and this inhibition is cancelled by Ca²⁺/calmodulin (CaM) formed during membrane depolarization. G-protein-gated K⁺ channel activity decreases on depolarization owing to the facilitation of GTPase-activating protein activity by RGS proteins and vice versa on hyperpolarization. The molecular mechanism responsible for this reciprocal control of RGS action by PtdIns(3,4,5)P₃ and Ca²⁺/CaM, however, has not been fully elucidated. Using lipid–protein co-sedimentation assay and surface plasmon resonance measurements, we show in the present study that the control of the GTPase-accelerating activity of the RGS4 protein is achieved through the competitive binding of PtdIns(3,4,5)P₃ and Ca²⁺/CaM within its RGS domain. Competitive binding occurs exclusively within the RGS domain and involves a cluster of positively charged residues located on the surface opposite to the Gα interaction site. In the RGS proteins conserving these residues, the reciprocal regulation by PtdIns(3,4,5)P₃ and Ca²⁺/CaM may be important for their physiological regulation of G-protein signalling.