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), se rves 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 her e 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 structu ral determinants that control the direction of P gamma effects on the RGS-G t alpha system are localized within the RGS domains. The addition of P gamm a caused an increase in the maximal stimulation of Gt alpha GTPase activity by RGS9d without affecting the EC50 value. Modulation of Gt alpha GTPase a ctivity by chimeric RGS16 and RGS9 proteins and P gamma has been investigat ed. This analysis suggests that in addition to the differences in primary s tructures, 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 an d RGS16 GAP activity, The RGS9 alpha 3-alpha 5 region constituted the minim al insertion of the RGS9 domain into RGS 16 that reversed the inhibitory ef fect 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 al pha. Our results and this model demonstrate that the mechanism of potentiat ion 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 .