Modulation of transducin GTPase activity by chimeric RGS16 and RGS9 regulators of G protein signaling and the effector molecule

RGS9, a member of the family of regulators of G protein signaling (RGS), serves as a GTPase-activating protein (GAP) for the transducin α-subunit (Gtα) in the vertebrate visual transduction cascade. The GAP activity of RGS9 is uniquely potentiated by the γ-subunit of the effector enzyme, cGMP- phosphodiesterase (Pγ). In contrast, Pγ attenuates the GAP effects of several other RGS proteins, including RGS16. We demonstrate here that the Pγ subunit exerts its effects on the GTPase activity of the Gtα-RGS complex via the C-terminal domain, Pγ-63-87. The structural determinants that control the direction of Pγ effects on the RGS-Gtα system are localized within the RGS domains. The addition of Pγ caused an increase in the maximal stimulation of Gtα GTPase activity by RGS9d without affecting the EC₅₀ value. Modulation of Gtα GTPase activity by chimeric RGS16 and RGS9 proteins and Pγ has been investigated. This analysis suggests that in addition to the differences in primary structures, the overall conformations of the RGS fold in RGS9 and RGS 16 are likely to be responsible for the opposite effects of Pγ on the RGS9 and RGS16 GAP activity. The RGS9 α3-α5 region constituted the minimal insertion of the RGS9 domain into RGS16 that reversed the inhibitory effect of Pγ. A model of the RGS9 complex with Gtα shows the α3-α5 helices in RGS9 facing the proximate Pγ binding site on Gtα. Our results and this model demonstrate that the mechanism of potentiation of RGS9 GAP activity by Pγ involves a more rigid stabilization of the Gtα switch regions when Gtα is bound to both RGS9 and Pγ.