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

RGS9, a member of the family of regulators of G protein signaling (RGS), serves as a GTPase-activating protein (GAP) for the transducin alpha-subunit (Gt alpha) in the vertebrate visual transduction cascade. The GAP activity of RGS9 is uniquely potentiated by the gamma-subunit of the effector enzyme, cGMP-phosphodiesterase (P gamma). In contrast, P gamma attenuates the GAP effects of several other RGS proteins, including RGS16. We demonstrate here that the P gamma subunit exerts its effects on the GTPase activity of the Gt alpha-RGS complex via the C-terminal domain, P gamma-63-87. The structural determinants that control the direction of P gamma effects on the RGS-Gt alpha system are localized within the RGS domains. The addition of P gamma caused an increase in the maximal stimulation of Gt alpha GTPase activity by RGS9d without affecting the EC50 value. Modulation of Gt alpha GTPase activity by chimeric RGS16 and RGS9 proteins and P gamma 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 RGS16 are likely to be responsible for the apposite effects of P gamma on the RGS9 and RGS16 GAP activity, The RGS9 alpha 3-alpha 5 region constituted the minimal insertion of the RGS9 domain into RGS 16 that reversed the inhibitory effect of P gamma. A model of the RGS9 complex with Gt alpha shows the alpha 3-alpha 5 helices in RGS9 being the proximate P gamma binding site on Gt alpha. Our results and this model demonstrate that the mechanism of potentiation of RGS9 GAP activity by P gamma involves a more rigid stabilization of the Gt alpha switch regions when Gt alpha is bound to both RGS9 and P gamma.